IL-17A/F cross-reactive monoclonal antibodies and methods of using the same

ABSTRACT

The present invention relates to antagonizing the activity of IL-17A, IL-17F and IL-23 using bispecific antibodies that comprise a binding entity that is cross-reactive for IL-17A and IL-17F and a binding entity that binds IL-23p19. The present invention relates to novel bispecific antibody formats and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is the National Stage filed under 35 U.S.C. §371of PCT Application No. PCT/US2013/041928, filed May 21, 2013, whichclaims the benefit of U.S. Patent Application Ser. No. 61/787,890, filedMar. 15, 2013, U.S. Patent Application Ser. No. 61/784,600, filed Mar.14, 2013, and U.S. Patent Application Ser. No. 61/650,286, filed May 22,2012, all of which are herein incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

Cytokines are soluble, small proteins that mediate a variety ofbiological effects, including the induction of immune cellproliferation, development, differentiation, and/or migration, as wellas the regulation of the growth and differentiation of many cell types(see, for example, Arai et al., Annu. Rev. Biochem. 59:783 (1990);Mosmann, Curr. Opin. Immunol. 3:311 (1991); Paul et al., Cell, 76:241(1994)). Cytokine-induced immune functions can also include aninflammatory response, characterized by a systemic or local accumulationof immune cells. Although they do have host-protective effects, theseimmune responses can produce pathological consequences when the responseinvolves excessive and/or chronic inflammation, as in autoimmunedisorders (such as multiple sclerosis) and cancer/neoplastic diseases(Oppenheim et al., eds., Cytokine Reference, Academic Press, San Diego,Calif. (2001); von Andrian et al., New Engl. J. Med., 343:1020 (2000);Davidson et al., New Engl. J. Med., 345:340 (2001); Lu et al., Mol.Cancer Res., 4:221 (2006); Dalgleish et al., Cancer Treat Res., 130:1(2006)).

IL-17A, IL-17F and IL-23 are cytokines involved in inflammation. IL-17Ainduces the production of inflammatory cytokines such as IL-1β, TNF-α,IL-6, and IL-23 by synovial fibroblasts, monocytes, and macrophages, allof which promote inflammation and Th17 development. IL-17A also inducesan array of chemokines, including CXCL-1, CXCL-2, CXCL-5, CXCL-8, CCL-2,and CCL-20, leading to recruitment of T cells, B cells, monocytes, andneutrophils. Lundy, S. K., Arthritis Res. Ther., 9:202 (2007). IL-17Fshares the greatest homology (55%) with IL-17A and is also aproinflammatory cytokine Both IL-17A and IL-17F are produced by Th17cells, whereas the other IL-17 family members, IL-17B, IL-17C, andIL-17D, are produced by non-T cell sources. IL-17A and IL-17F can existas IL-17A homodimers and IL-17F homodimers or as IL-17A/F heterodimers.Liang, S. C. et al., J. Immunol., 179:7791-7799 (2007). IL-17A isincreased in rheumatoid arthritis sera and synovial fluid, and ispresent in the T-cell rich areas of the synovium. Shahrara, S.,Arthritis Res. Ther., 10:R93 (2005). IL-17A can also orchestrate boneand cartilage damage. An effective blockade of IL-17 will need toneutralize IL-17A homodimers, IL-17F homodimers and IL-17A/Fheterodimers.

IL-23 is a type-1 heterodimer, comprising a 19 kilodalton (kD) fourfoldhelical core a subunit (IL-23p19), disulfide linked to an additional 40kD distinct β subunit (IL-12p40). IL-23 is a key cytokine in bridgingthe innate and adaptive arms of the immune response; it is producedearly in response to an antigen challenge, and is essential for drivingearly local immune responses. Furthermore, IL-23 plays a central role inthe activation of NK cells, the enhancement of T cell proliferation andthe regulation of antibody production. IL-23 also regulatespro-inflammatory cytokines (e.g., IFN-γ), which are important incell-mediated immunity against intracellular pathogens. Recent reportshave indicated that in humans increased amounts of IL-23 have beenassociated with several autoimmune diseases including rheumatoidarthritis (RA), Lyme arthritis, inflammatory bowel disease (IBD),Crohn's disease (CD), psoriasis and multiple sclerosis (MS). IL-23p19knock-out mice were resistant to autoimmune encephalomyelitis (EAE),collagen-induced arthritis (CIA) and central nervous system autoimmuneinduction. IL-23 is not essential for the development of human Th17cells, but appears to be required for their survival and/or expansion.Paradowska-Gorycka, A., Scandinavian Journal of Immunology, 71:134-145(2010). Genetic studies revealed an association between IL-23 receptorgenes and susceptibility to several autoimmune diseases including CD, RAand Graves' ophthalmopathy. The IL-23-Th17 axis is crucial to autoimmunedisease development. Leng et al., Archives of Medical Research,41:221-225 (2010).

The demonstrated activities of IL-17A, IL-17F and IL-23p19 in mediatingand promoting several autoimmune diseases illustrate the clinicalpotential of, and need for, molecules which can antagonize thesetargets. The present invention, as set forth herein, meets these andother needs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a whole antibody and its modularcomponents.

FIG. 2 depicts a model of a bispecific antibody designated biAbFabLwhich contains a whole antibody with a C-terminal Fab unit of the secondarm of the bispecific antibody attached via a linker, and which utilizesa common light chain.

FIG. 3 depicts a model of a bispecific antibody designated taFab whichcontains a whole antibody with an N-terminal Fab unit of the second armof the bispecific antibody attached via a linker. As with the heavychain portion, there are two light chains for each arm of the bispecificattached via a linker.

FIG. 4 depicts a model of a bispecific antibody designated HeterodimericFc, that resembles a traditional antibody, however, contains twodifferent heavy chains which associate through an electrostaticcomplementarity association in the C_(H3) region. The Heterodimeric Fcutilizes a common light chain.

FIG. 5 depicts a model of a bispecific antibody designated VCVFc whichcontains a whole antibody with a Fv unit of the second arm of thebispecific antibody inserted between the Fab region and the hinge vialinkers.

FIG. 6 depicts a model of a bispecific antibody designated VCDFc whichcontains a whole antibody with a single domain antibody for the secondarm of the bispecific antibody inserted between the Fab region and thehinge via linkers.

FIG. 7 illustrates the ELISA results showing strong antibody binding toIL-23p19 and lack of cross reactivity to IL-12.

FIG. 8 illustrates the potent neutralization of IL-23 signaling asobserved in the kit225 assay.

FIG. 9 shows the Biacore results of 7B7, STELARA® (ustekinumab, ananti-IL-23p40 antibody) and human IL-23 receptor ability to bind thevarious IL-23p19 alanine shaved mutants, wild-type and purifiedwild-type L-23p19 (positive control) and a negative control. 7B7 mAbbinding is shown in the left column, STELARA® is shown in the centercolumn and hIL-23R-Fc binding is shown in the right column. The fourmutants shown with the star were chosen for scale-up to confirm theseresults.

FIG. 10 shows a Biacore kinetic analysis of IL-23p19 antibody binding tothe IL-23 alanine mutants.

FIG. 11 schematically illustrates the process used to identify andselect the 7B7 antibody (anti-IL-23p19).

FIG. 12 schematically illustrates the clinical disease scores over timein the marmoset EAE model.

FIG. 13 schematically illustrates the MRI lesion score in the marmosetEAE model.

FIG. 14 schematically illustrates the MRI optic nerve score in themarmoset EAE model.

FIG. 15 shows the overlay of all four structures of the Fab with IL-17Aor IL-17F, aligned by the interleukin.

FIG. 16 graphically shows the cell functional activity of the IL-17Amutants.

FIG. 17 graphically shows the cell functional activity of the IL-17Fmutants.

FIG. 18 is a schematic overly of the 9 nM IL-17A mutants binding BiAb3demonstrating the accelerated off-rate of mutants containing Y108Amutation and combinations thereof.

FIG. 19 shows the computational energetic analysis of IL-17A mutantsbinding to BiAb3.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, in one embodiment, bispecific antibodiescomprising an IL-17A/F binding entity that binds to IL-17A and IL-17Fand an IL-23 binding entity that binds to IL-23 via p19. The inventionalso includes isolated nucleic acids encoding the heavy chains and lightchains of the bispecific antibodies of the invention, as well as vectorscomprising the nucleic acids, host cells comprising the vectors, andmethods of making and using the bispecific antibodies.

In other embodiments, the present invention provides compositionscomprising the bispecific antibodies and kits comprising the bispecificantibodies, as well as articles of manufacture comprising the bispecificantibodies.

The bispecific antibodies of the present invention are useful for theinhibition of proinflammatory cytokines, e.g., IL-17A, IL-17F andIL-23p19. The antibodies can be used to reduce, limit, neutralize, orblock the proinflammatory effects of the IL-17A homodimer, the IL-17Fhomodimer, or the IL-17A/F heterodimer. Likewise, the antibodies can beused to reduce, limit, neutralize, or block the pro-cancerous effects ofthe IL-17A homodimer, the IL-17F homodimer, or the IL-17A/F heterodimer.In such cases, the anti-IL-23p19 portion of the antibody is used toreduce, limit, neutralize, or block production of new T cells that wouldproduce IL-17A and/or IL-17F, including homodimers and heterodimers. Thebispecific antibodies described herein can be used to treat inflammatorydisorders and autoimmune diseases, such as multiple sclerosis (e.g.,relapsing-remitting multiple sclerosis, secondary-progressive multiplesclerosis, primary-progressive multiple sclerosis andprogressive-relapsing multiple sclerosis), inflammatory bowel disease,psoriasis, systemic sclerosis, systemic lupus erythematosus,antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV)and giant cell arteritis. The bispecific antibodies described herein canalso be used to treat cancer, including angiogenesis. For instance, thebispecific antibodies as described herein can be used to treatmultiple-myeloma-induced lytic bone disease (Sotomayor, E. M., Blood,116(18):3380-3382 (2010)).

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

Unless otherwise specified, “a”, “an”, “the”, and “at least one” areused interchangeably and mean one or more than one.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.Thus, as used herein, the term “antibody” or “antibody peptide(s)”refers to an intact antibody, or an antigen-binding fragment thereofthat competes with the intact antibody for specific binding and includeschimeric, humanized, fully human, and bispecific antibodies. In certainembodiments, antigen-binding fragments are produced, for example, byrecombinant DNA techniques. In additional embodiments, antigen-bindingfragments are produced by enzymatic or chemical cleavage of intactantibodies. Antigen-binding fragments include, but are not limited to,Fab, Fab′, F(ab)², F(ab′)², Fv, and single-chain antibodies.

The term “isolated antibody” as used herein refers to an antibody thathas been identified and separated and/or recovered from a component ofits natural environment. Contaminant components of its naturalenvironment are materials which would interfere with diagnostic ortherapeutic uses for the antibody, and may include enzymes, hormones,and other proteinaceous or nonproteinaceous solutes. In preferredembodiments, the antibody will be purified (1) to greater than 95% byweight of antibody as determined by the Lowry method, and mostpreferably more than 99% by weight, (2) to a degree sufficient to obtainat least 15 residues of N-terminal or internal amino acid sequence byuse of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGEunder reducing or nonreducing conditions using Coomassie blue or,preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

The term “agonist” refers to any compound including a protein,polypeptide, peptide, antibody, antibody fragment, large molecule, orsmall molecule (less than 10 kD), that increases the activity,activation or function of another molecule.

The term “antagonist” refers to any compound including a protein,polypeptide, peptide, antibody, antibody fragment, large molecule, orsmall molecule (less than 10 kD), that decreases the activity,activation or function of another molecule.

The term “bind(ing) of a polypeptide” includes, but is not limited to,the binding of a ligand polypeptide of the present invention to areceptor; the binding of a receptor polypeptide of the present inventionto a ligand; the binding of an antibody of the present invention to anantigen or epitope; the binding of an antigen or epitope of the presentinvention to an antibody; the binding of an antibody of the presentinvention to an anti-idiotypic antibody; the binding of ananti-idiotypic antibody of the present invention to a ligand; thebinding of an anti-idiotypic antibody of the present invention to areceptor; the binding of an anti-anti-idiotypic antibody of the presentinvention to a ligand, receptor or antibody, etc.

A “bispecific” or “bifunctional” antibody is a hybrid antibody havingtwo different heavy/light chain pairs and two different binding sites.Bispecific antibodies may be produced by a variety of methods including,but not limited to, fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990);Kostelny et al., J. Immunol., 148:1547-1553 (1992).

As used herein, the term “epitope” refers to the portion of an antigento which an antibody specifically binds. Thus, the term “epitope”includes any protein determinant capable of specific binding to animmunoglobulin or T-cell receptor. Epitopic determinants usually consistof chemically active surface groupings of molecules such as amino acidsor sugar side chains and usually have specific three dimensionalstructural characteristics, as well as specific charge characteristics.More specifically, the term “IL-17 epitope”, “IL-23 epitope” and/or“IL-23/p19 epitope” as used herein refers to a portion of thecorresponding polypeptide having antigenic or immunogenic activity in ananimal, preferably in a mammal, and most preferably in a mouse or ahuman. An epitope having immunogenic activity is a portion of, forexample, an IL-17A or IL-17F or IL-23/p19 polypeptide that elicits anantibody response in an animal. An epitope having antigenic activity isa portion of, for example, an IL-17A or IL-17F or IL-23/p19 polypeptideto which an antibody immunospecifically binds as determined by anymethod well known in the art, for example, by immunoassays, proteasedigest, crystallography or H/D-Exchange. Antigenic epitopes need notnecessarily be immunogenic. Such epitopes can be linear in nature or canbe a discontinuous epitope. Thus, as used herein, the term“conformational epitope” refers to a discontinuous epitope formed by aspatial relationship between amino acids of an antigen other than anunbroken series of amino acids.

As used herein, the term “immunoglobulin” refers to a protein consistingof one or more polypeptides substantially encoded by immunoglobulingenes. One form of immunoglobulin constitutes the basic structural unitof an antibody. This form is a tetramer and consists of two identicalpairs of immunoglobulin chains, each pair having one light and one heavychain. In each pair, the light and heavy chain variable regions aretogether responsible for binding to an antigen, and the constant regionsare responsible for the antibody effector functions.

Full-length immunoglobulin “light chains” (about 25 Kd or about 214amino acids) are encoded by a variable region gene at the NH2-terminus(about 110 amino acids) and a kappa or lambda constant region gene atthe COOH-terminus. Full-length immunoglobulin “heavy chains” (about 50Kd or about 446 amino acids), are similarly encoded by a variable regiongene (about 116 amino acids) and one of the other aforementionedconstant region genes (about 330 amino acids). Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG (such as IgG1, IgG2, IgG3 and IgG4), IgM, IgA,IgD and IgE, respectively. Within light and heavy chains, the variableand constant regions are joined by a “J” region of about 12 or moreamino acids, with the heavy chain also including a “D” region of about10 more amino acids. (See generally, Fundamental Immunology (Paul, W.,ed., 2nd Edition, Raven Press, NY (1989)), Chapter 7 (incorporated byreference in its entirety for all purposes).

An immunoglobulin light or heavy chain variable region consists of a“framework” region interrupted by three hypervariable regions. Thus, theterm “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen binding. The hypervariableregion comprises amino acid residues from a “Complementarity DeterminingRegion” or “CDR” (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Edition, Public Health Service, National Institutes ofHealth, Bethesda, Md. (1991)) and/or those residues from a“hypervariable loop” (Chothia et al., J. Mol. Biol. 196: 901-917 (1987))(both of which are incorporated herein by reference). “Framework Region”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined. The sequences of theframework regions of different light or heavy chains are relativelyconserved within a species. Thus, a “human framework region” is aframework region that is substantially identical (about 85% or more,usually 90-95% or more) to the framework region of a naturally occurringhuman immunoglobulin. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDR's. The CDR's are primarilyresponsible for binding to an epitope of an antigen. Accordingly, theterm “humanized” immunoglobulin refers to an immunoglobulin comprising ahuman framework region and one or more CDR's from a non-human (usually amouse or rat) immunoglobulin. The non-human immunoglobulin providing theCDR's is called the “donor” and the human immunoglobulin providing theframework is called the “acceptor”. Constant regions need not bepresent, but if they are, they must be substantially identical to humanimmunoglobulin constant regions, i.e., at least about 85-90%, preferablyabout 95% or more identical. Hence, all parts of a humanizedimmunoglobulin, except possibly the CDR's, are substantially identicalto corresponding parts of natural human immunoglobulin sequences.Further, one or more residues in the human framework region may be backmutated to the parental sequence to retain optimal antigen-bindingaffinity and specificity. In this way, certain framework residues fromthe non-human parent antibody are retained in the humanized antibody inorder to retain the binding properties of the parent antibody whileminimizing its immunogenicity. The term “human framework region” as usedherein includes regions with such back mutations. A “humanized antibody”is an antibody comprising a humanized light chain and a humanized heavychain immunoglobulin. For example, a humanized antibody would notencompass a typical chimeric antibody as defined above, e.g., becausethe entire variable region of a chimeric antibody is non-human.

The term “humanized” immunoglobulin refers to an immunoglobulincomprising a human framework region and one or more CDR's from anon-human, e.g., mouse, rat or rabbit, immunoglobulin. The non-humanimmunoglobulin providing the CDR's is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor”.Constant regions need not be present, but if they are, they must besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-90%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDR's andpossibly a few back-mutated amino acid residues in the framework region(e.g., 1-15 residues), are substantially identical to correspondingparts of natural human immunoglobulin sequences. A “humanized antibody”is an antibody comprising a humanized light chain and a humanized heavychain immunoglobulin. For example, a humanized antibody would notencompass a typical chimeric antibody as defined above, e.g., becausethe entire variable region of a chimeric antibody is non-human.

As used herein, the term “human antibody” includes an antibody that hasan amino acid sequence of a human immunoglobulin and includes antibodiesisolated from human immunoglobulin libraries or from animals transgenicfor one or more human immunoglobulin and that do not express endogenousimmunoglobulins, as described, for example, by Kucherlapati et al. inU.S. Pat. No. 5,939,598.

The term “genetically altered antibodies” means antibodies wherein theamino acid sequence has been varied from that of a native antibody.Because of the relevance of recombinant DNA techniques in the generationof antibodies, one need not be confined to the sequences of amino acidsfound in natural antibodies; antibodies can be redesigned to obtaindesired characteristics. The possible variations are many and range fromthe changing of just one or a few amino acids to the complete redesignof, for example, the variable and/or constant region. Changes in theconstant region will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with membranesand other effector functions. Changes in the variable region will bemade in order to improve the antigen binding characteristics.

A “Fab fragment” is comprised of one light chain and the C_(H1) andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” contains one light chain and one heavy chain thatcontains more of the constant region, between the C_(H1) and C_(H2)domains, such that an interchain disulfide bond can be formed betweentwo heavy chains to form a F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H1) andC_(H2) domains, such that an interchain disulfide bond is formed betweentwo heavy chains.

A “Fv fragment” contains the variable regions from both heavy and lightchains but lacks the constant regions.

A “single domain antibody” is an antibody fragment consisting of asingle domain Fv unit, e.g., V_(H) or V_(L). Like a whole antibody, itis able to bind selectively to a specific antigen. With a molecularweight of only 12-15 kDa, single-domain antibodies are much smaller thancommon antibodies (150-160 kDa) which are composed of two heavy proteinchains and two light chains, and even smaller than Fab fragments (˜50kDa, one light chain and half a heavy chain) and single-chain variablefragments (˜25 kDa, two variable domains, one from a light and one froma heavy chain). The first single-domain antibodies were engineered fromheavy-chain antibodies found in camelids. Although most research intosingle-domain antibodies is currently based on heavy chain variabledomains, light chain variable domains and nanobodies derived from lightchains have also been shown to bind specifically to target epitopes.

The term “monoclonal antibody” or “mAb” or “MAb” or “Mab” or “mab” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” or “mAb” or “MAb” or “Mab” or“mab” refer to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids”, whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol., 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol., 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem., 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem., 269:25728 (1994)), SP1, cAMP response elementbinding protein (CREB; Loeken, Gene Expr., 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th Edition, The Benjamin/Cummings Publishing Company, Inc.(1987), and Lemaigre et al., Biochem. J., 303:1 (1994)). If a promoteris an inducible promoter, then the rate of transcription increases inresponse to an inducing agent. In contrast, the rate of transcription isnot regulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific”,“tissue-specific”, or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that produces anantagonist of the present invention from an expression vector. Incontrast, such an antagonist can be produced by a cell that is a“natural source” of said antagonist, and that lacks an expressionvector.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes. Forexample, a fusion protein can comprise at least part of a IL-17RApolypeptide fused with a polypeptide that binds an affinity matrix. Sucha fusion protein provides a means to isolate large quantities of IL-17Ausing affinity chromatography.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. Receptors can be membrane bound,cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormonereceptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor,growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor,erythropoietin receptor and IL-6 receptor). Membrane-bound receptors arecharacterized by a multi-domain structure comprising an extracellularligand-binding domain and an intracellular effector domain that istypically involved in signal transduction. In certain membrane-boundreceptors, the extracellular ligand-binding domain and the intracellulareffector domain are located in separate polypeptides that comprise thecomplete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity ofless than 10⁹ M⁻¹.

As used herein, a “therapeutic agent” is a molecule or atom which isconjugated to an antibody moiety to produce a conjugate which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A “detectable label” is a molecule or atom which can be conjugated to anantibody moiety to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, or other marker moieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J., 4:1075 (1985);Nilsson et al., Methods Enzymol., 198:3 (1991)), glutathione Stransferase (Smith et al., Gene, 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA, 82:7952 (1985)),substance P, FLAG® peptide (Hopp et al., Biotechnology, 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification, 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

An “IL-17A binding entity” is a binding entity, such as an antibody,that specifically binds to IL-17A in its homodimeric form(IL-17A/IL-17A) and in its heterodimeric form (IL-17A/IL-17F).

An “IL-17F binding entity” is a binding entity, such as an antibody,that specifically binds to IL-17F in its homodimeric form(IL-17F/IL-17F) and in its heterodimeric form (IL-17A/IL-17F).

An “IL-17A/F binding entity” is a binding entity, such as an antibody,that specifically binds to IL-17A and IL-17F by recognizing and bindingto the same or similar epitope, e.g., continuous or discontinuousepitope, shared by IL-17A and IL-17F. The IL-17A/F binding entity bindsto the IL-17A homodimer, IL-17F homodimer and IL-17A/IL-17F heterodimer.

One problem is that IL-17A, IL-17F and IL-23p19 are overexpressed in andimplicated in the cause and/or sustainability of several inflammatoryand/or autoimmune disorders. One solution several embodiments of thepresent invention provide is to inhibit or reduce the ability of thesecytokines to cell-signal by administering a bispecific antibody whichbinds to each cytokine and inhibits or reduces each cytokine, e.g.,homodimeric or heterodimeric form, from signaling through their cognatereceptor.

Another problem is that in creating a bispecific IL-17A/F and IL-23p19bispecific antibody, e.g, biAb3, difficulties were encountered togenerate a bispecific antibody which had high affinity to IL-17A and wasan effective neutralizer, e.g., IC50, of IL-17A. The mouse parentantibody (e.g, chimeric 339.15, 339.15.3.5, 339.15.5.3 or 339.15.3.6) asshown in Table 1 had an IL-17A/F IC50 of 0.30 nM and an IL-17F IC50 of0.26 nM, but only had an IL-17A IC50 of 11 nM. The antibodies ability toinhibit or neutralize IL-17A from signaling needed to be enhanced. Asshown in Table 3, mouse parent chimeric 339.15 and humanized parent339.134 had a similar binding affinity towards IL-17A. Furthermore, asshown in Table 8, humanized parent 339.134 ability to inhibit IL-17A(IC50 of 1.3 nM) was still significantly reduced as compared to itsability to inhibit IL-17A/F (IC50 of 0.27 nM) and IL-17F (IC50 of 0.24nM). This problem was surprisingly overcome by utilizing the light chainof the IL-23p19 antibody of biAb3 (SEQ ID NO:17). When the IL-23p19light chain was paired with the humanized heavy chain of 339.134, theresulting monoclonal antibody significantly increased its ability toinhibit IL-17A from signaling (see Table 9) and significantly increasedits affinity for IL-17A (see Table 10). The critical residue in the nowshared IL-23p19/IL-17A/F light chain of, for example, biAb3, that mayhave provided this enhanced affinity and neutralization ability may beY108 or Tyr108 of SEQ ID NO:2 as evidence by the X-ray crystallographyand alanine mutant studies of Example 9.

In one embodiment, the present invention provides bispecific antibodies,antibodies and antigen-binding fragments thereof. The bispecificantibodies of the invention comprise an IL-17A binding entity that bindsto IL-17A and an IL-23 binding entity that binds to IL-23 via p19. Inanother aspect, the bispecific antibodies of the invention comprise anIL-17F binding entity that binds to IL-17F and a IL-23 binding entitythat binds to IL-23 via p19. In another aspect, the bispecificantibodies of the invention comprise an IL-17A/F binding entity thatbinds to IL-17A and IL-17F and a IL-23 binding entity that binds toIL-23 via p19. The binding entity that binds to IL-23 via p19 isreferred to hereinafter as a binding entity that binds to IL-23 or an“IL-23 binding entity”. The polynucleotide sequence of the human IL-17Ais shown in SEQ ID NO:1 and the corresponding polypeptide sequence isshown in SEQ ID NO:2. The signal sequence of the IL-17A polypeptide isamino acid residues 1-23 of SEQ ID NO:2. Thus, amino acid residues24-155 of SEQ ID NO:2 constitute the mature IL-17A polypeptide.Antibodies (and antigen-binding fragments thereof) and bispecificantibodies disclosed herein that bind to IL-17A bind to the matureIL-17A polypeptide (amino acid residues 24-155 of SEQ ID NO:2). Thepolynucleotide sequence of the human IL-17F is shown in SEQ ID NO:3 andthe corresponding polypeptide sequence is shown in SEQ ID NO:4. Thesignal sequence of the IL-17F polypeptide is amino acid residues 1-30 ofSEQ ID NO:4. Thus, amino acid residues 31-163 of SEQ ID NO:4 constitutethe mature IL-17F polypeptide. Antibodies (and antigen-binding fragmentsthereof) and bispecific antibodies disclosed herein that bind to IL-17Fbind to the mature IL-17F polypeptide (amino acid residues 31-163 of SEQID NO:4). The polynucleotide sequence of the human p19 subunit of IL-23is shown in SEQ ID NO:5 and the corresponding polypeptide sequence isshown in SEQ ID NO:6. The signal sequence of the IL-23p19 polypeptide isamino acid residues 1-19 of SEQ ID NO:6. Thus, amino acid residues20-189 of SEQ ID NO:6 constitute the mature IL-23p19 polypeptide.Antibodies (and antigen-binding fragments thereof) and bispecificantibodies disclosed herein that bind to IL-23p19 bind to the matureIL-23p19 polypeptide (amino acid residues 20-189 of SEQ ID NO:6).

In one aspect of the invention, the IL-17A/F binding entity comprises anantibody, i.e., two pairs of immunoglobulin chains, each pair having onelight and one heavy chain, and the IL-23 binding entity comprises twoFab fragments each comprising a light chain and the C_(H1) and variableregions of a heavy chain, and the Fab fragments of the IL-23 bindingentity are linked to the C-termini of the heavy chains (Fc) of theIL-17A/F binding entity. This bispecific antibody format is referred toherein as biAbFabL (see FIG. 2). In another embodiment, each of thelight chain and the C_(H1) and variable regions of the heavy chaincomprising the Fab fragments of the IL-23 binding entity are linked tothe N-termini of the light chains and heavy chains, respectively, of theIL-17A/F binding entity. This bispecific antibody format is referred toherein as taFab (see FIG. 3).

In another aspect of the invention, the IL-23 binding entity comprisesan antibody, i.e., two pairs of immunoglobulin chains, each pair havingone light and one heavy chain, and the IL-17A/F binding entity comprisestwo Fab fragments each comprising a light chain and the C_(H1) andvariable regions of a heavy chain, and the Fab fragments of the IL-17A/Fbinding entity are linked to the C-termini of the heavy chains (Fc) ofthe IL-23 binding entity. This bispecific antibody format is referred toherein as biAbFabL (See FIG. 2). In another embodiment, each of thelight chain and the C_(H1) and variable regions of the heavy chaincomprising the Fab fragments of the IL-17A/F binding entity are linkedto the N-termini of the light chain and heavy chain, respectively, ofthe IL-23 binding entity. This bispecific antibody format is referred toherein as taFab (see FIG. 3).

In another aspect of the invention, the IL-23 binding entity comprises alight chain and an IL-23 heavy chain and the IL-17A/F binding entitycomprises a light chain and an IL-17A/F heavy chain. This bispecificantibody resembles a traditional antibody except that it comprises twodifferent heavy chains that associate through an electrostaticcomplementarity association in the C_(H3) regions. It utilizes a commonlight chain. This bispecific antibody format is referred to herein asHeterodimeric Fc (see FIG. 4).

In another embodiment, the present invention provides bispecificantibodies comprising a first binding entity comprising an antibody,i.e., two pairs of immunoglobulin chains, each pair having one lightchain and one heavy chain, and a second binding entity comprising an Fvunit, i.e., the variable domains from a heavy and a light chain, and inwhich the second binding entity comprising the Fv unit is positionedbetween the Fab region and the hinge of the first binding entity asshown in FIG. 5. The Fv unit is linked to the Fab region of the firstbinding entity by linker molecules. More specifically, the Fv unitcomprises a variable light domain which is linked to the light chainconstant region of the Fab fragment, and a variable heavy domain whichis linked to the C_(H1) region of the Fab fragment. This bispecificantibody format is referred to herein as VCVFc. The first binding entityand second binding entity of a VCVFc do not have to share a common lightchain, while the first binding entity and second binding entity of abiAbFabL do have to share a common light chain. In one aspect of thisembodiment of the invention, the first binding entity specifically bindsa lymphocyte antigen, cytokine, cytokine receptor, growth factor, growthfactor receptor, interleukin (e.g., IL-17A, IL-17F, IL-17A/F and IL-23)or interleukin receptor and the second binding entity specifically bindsa lymphocyte antigen, cytokine, cytokine receptor, growth factor, growthfactor receptor, interleukin (e.g., IL-17A, IL-17F, IL-17A/F and IL-23)or interleukin receptor. In another aspect of this embodiment of theinvention, the first binding entity is an IL-17A/F binding entity andthe second binding entity is an IL-23 binding entity. In another aspectof this embodiment of the invention, the first binding entity is anIL-23 binding entity and the second binding entity is an IL-17A/Fbinding entity.

In another embodiment, the present invention provides bispecificantibodies comprising a first binding entity comprising an antibody,i.e., two pairs of immunoglobulin chains, each pair having one lightchain and one heavy chain, and a second binding entity comprising asingle domain antibody. This bispecific antibody format is referred toherein as VCDFc. An illustration of a VCDFc bispecific antibody is shownin FIG. 6. The second binding entity comprising the single domainantibody is positioned between the Fab region, more specifically theC_(H1) region of the Fab fragment, and the hinge of the first bindingentity. The single domain antibody is linked to the C_(H1) region of theFab of the first binding entity by linker molecules (for example, butnot limited to, 10 mer G₄S, which is represented by the equation (G₄S)₂,or SSASTKGPS (SEQ ID NO:86)). In one aspect of this embodiment of theinvention, the first binding entity specifically binds a lymphocyteantigen, cytokine, cytokine receptor, growth factor, growth factorreceptor, interleukin (e.g., IL-17A, IL-17F, IL-17A/F and IL-23) orinterleukin receptor and the second binding entity specifically binds alymphocyte antigen, cytokine, cytokine receptor, growth factor, growthfactor receptor, interleukin (e.g., IL-17A, IL-17F, IL-17A/F and IL-23)or interleukin receptor. In one aspect of this embodiment of theinvention, the first binding entity is an IL-23 binding entity and thesecond binding entity is an IL-17A/F binding entity. In another aspectof this embodiment of the invention, the first binding entity is anIL-17A/F binding entity and the second binding entity is an IL-23binding entity.

The amino acid sequences of the binding entities are preferably basedupon the sequences of human and/or humanized monoclonal antibodiesagainst a lymphocyte antigen, cytokine, cytokine receptor, growthfactor, growth factor receptor, interleukin (e.g., IL-17A, IL-17F,IL-17A/F and IL-23) or interleukin receptor.

In one embodiment of the foregoing aspects of the invention, the lightchains of the IL-17A/F binding entity and the IL-23 binding entity ofthe bispecific antibody each comprise a variable domain comprising aCDR1 having the amino acid sequence of SEQ ID NO:22, a CDR2 having theamino acid sequence of SEQ ID NO:23, and a CDR3 having the sequence ofSEQ ID NO:24. In another embodiment, the light chains of the IL-17A/Fbinding entity and the IL-23 binding entity each comprise a variabledomain comprising the amino acid sequence of SEQ ID NO:9. In anotherembodiment, the light chains of the IL-17A/F binding entity and theIL-23 binding entity each comprise a constant domain comprising theamino acid sequence of SEQ ID NO:10. In another embodiment, the lightchains of the IL-17A/F binding entity and the IL-23 binding entity eachcomprise a variable domain comprising the amino acid sequence of SEQ IDNO:9 and a constant domain comprising the amino acid sequence of SEQ IDNO:10.

In another embodiment of the foregoing aspects of the invention, theheavy chain of the IL-17A/F binding entity of the bispecific antibodycomprises a variable domain comprising a CDR1 having the amino acidsequence of SEQ ID NO:25, a CDR2 having the amino acid sequence of SEQID NO:26, and a CDR3 having the amino acid sequence of SEQ ID NO:27. Inanother embodiment, the heavy chain of the IL-17A/F binding entitycomprises a variable domain comprising the amino acid sequence of SEQ IDNO:13. In another embodiment, when the IL-17A/F binding entity comprisesan antibody, the heavy chain constant domain comprises the amino acidsequence of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:127 or SEQ ID NO:128.In another embodiment, when the IL-17A/F binding entity comprises a Fabfragment, the C_(H1) region of the heavy chain comprises the amino acidsequence of SEQ ID NO:14 or SEQ ID NO:15.

In another embodiment of the foregoing aspects of the invention, theIL-17A/F binding entity of the bispecific antibody comprises a heavychain variable domain having at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% sequence identity with the amino acid sequenceof SEQ ID NO:13. Optionally, all of the substitutions, additions ordeletions are within the framework region of the heavy chain variabledomain. Optionally, the IL-17A/F binding entity of the bispecificantibody comprises a heavy chain variable domain having at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% sequence identitywith the amino acid sequence of SEQ ID NO:13, wherein the variabledomain comprises a CDR1 having the amino acid sequence of SEQ ID NO:25,a CDR2 having the amino acid sequence of SEQ ID NO:26, and a CDR3 havingthe amino acid sequence of SEQ ID NO:27. Optionally, the heavy chainvariable domain comprises the amino acid sequence of SEQ ID NO:13.Optionally, the three IL-17A/F heavy chain variable domain CDRs includea CDR1 region comprising an amino acid sequence having at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% sequence identitywith the amino acid sequence of SEQ ID NO:25; a CDR2 region comprisingan amino acid sequence having at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% sequence identity with the amino acid sequenceof SEQ ID NO:26; and a CDR3 region comprising an amino acid sequencehaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% sequence identity with the amino acid sequence of SEQ ID NO:27.Optionally, the IL-17A/F heavy chain variable domain CDR1 has the aminoacid sequence of SEQ ID NO:25; the heavy chain variable domain CDR2 hasthe amino acid sequence of SEQ ID NO:26; and the heavy chain variabledomain CDR3 has the amino acid sequence of SEQ ID NO:27. The IL-17A/Fand/or IL-23p19 binding entity comprises a light chain variable domainhaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% sequence identity with the amino acid sequence of SEQ ID NO:9.Optionally, all of the substitutions, additions or deletions are withinthe framework region of the light chain variable domain. Optionally, theIL-17A/F and/or IL-23p19 binding entity of the bispecific antibodycomprises a light chain variable domain having at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% sequence identity with theamino acid sequence of SEQ ID NO:9, wherein the variable domaincomprises a CDR1 having the amino acid sequence of SEQ ID NO:22, a CDR2having the amino acid sequence of SEQ ID NO:23, and a CDR3 having theamino acid sequence of SEQ ID NO:24. Optionally, the light chainvariable domain comprises the amino acid sequence of SEQ ID NO:9.Optionally, the three IL-17A/F and/or IL-23p19 light chain variabledomain CDRs include a CDR1 region comprising an amino acid sequencehaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% sequence identity with the amino acid sequence of SEQ ID NO:22; aCDR2 region comprising an amino acid sequence having at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% sequence identitywith the amino acid sequence of SEQ ID NO:23; and a CDR3 regioncomprising an amino acid sequence having at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% sequence identity with the aminoacid sequence of SEQ ID NO:24. Optionally, the IL-17A/F and/or IL-23p19light chain variable domain CDR1 has the amino acid sequence of SEQ IDNO:22; the IL-17A/F and/or IL-23p19 light chain variable domain CDR2 hasthe amino acid sequence of SEQ ID NO:23; and the IL-17A/F and/orIL-23p19 light chain variable domain CDR3 has the amino acid sequence ofSEQ ID NO:24. The IL-23p19 binding entity comprises a heavy chainvariable domain having at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% sequence identity with the amino acid sequenceof SEQ ID NO:7. Optionally, all of the substitutions, additions ordeletions are within the framework region of the IL-23p19 heavy chainvariable domain. Optionally, the IL-23p19 binding entity of thebispecific antibody comprises a heavy chain variable domain having atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity with the amino acid sequence of SEQ ID NO:7, whereinthe variable domain comprises a CDR1 having the amino acid sequence ofSEQ ID NO:19, a CDR2 having the amino acid sequence of SEQ ID NO:20, anda CDR3 having the amino acid sequence of SEQ ID NO:21. Optionally, theIL-23p19 heavy chain variable domain comprises the amino acid sequenceof SEQ ID NO:7. Optionally, the three IL-23p19 heavy chain variabledomain CDRs include a CDR1 region comprising an amino acid sequencehaving at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% sequence identity with the amino acid sequence of SEQ ID NO:19; aCDR2 region comprising an amino acid sequence having at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% sequence identitywith the amino acid sequence of SEQ ID NO:20; and a CDR3 regioncomprising an amino acid sequence having at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% sequence identity with the aminoacid sequence of SEQ ID NO:21. Optionally, the IL-23p19 heavy chainvariable domain CDR1 has the amino acid sequence of SEQ ID NO:19; theheavy chain variable domain CDR2 has the amino acid sequence of SEQ IDNO:20; and the heavy chain variable domain CDR3 has the amino acidsequence of SEQ ID NO:21.

In another embodiment of the foregoing aspects of the invention, theheavy chain of the IL-23 binding entity of the bispecific antibodycomprises a variable domain comprising a CDR1 having the amino acidsequence of SEQ ID NO:19, a CDR2 having the amino acid sequence of SEQID NO:20, and a CDR3 having the amino acid sequence of SEQ ID NO:21. Inanother embodiment, the heavy chain of the IL-23 binding entitycomprises a variable domain comprising the amino acid sequence of SEQ IDNO:7. In another embodiment, when the IL-23 binding entity comprises anantibody, the heavy chain constant domain comprises the amino acidsequence of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:127 or SEQ ID NO:128.In some embodiments, the C-terminal lysine of SEQ ID NO:8 has beencleaved, and so the heavy chain constant domain comprises the amino acidsequence of residues 1-326 of SEQ ID NO:8. In another embodiment, whenthe IL-23 binding entity comprises a Fab fragment, the C_(H1) region ofthe heavy chain comprises the amino acid sequence of SEQ ID NO:14 or SEQID NO:15.

In another embodiment of the foregoing aspects of the invention, whenthe IL-23 binding entity or IL-17A/F binding entity of the bispecificantibody is an Fv unit, the variable domain of the light chain comprisesa CDR1 having the amino acid sequence of SEQ ID NO:22, a CDR2 having theamino acid sequence of SEQ ID NO:23, and a CDR3 having the sequence ofSEQ ID NO:24. In another embodiment, the light chains of the IL-17A/Fbinding entity and the IL-23 binding entity each comprise a variabledomain comprising the amino acid sequence of SEQ ID NO:9.

In another embodiment of the foregoing aspects of the invention, whenthe IL-17A/F binding entity of the bispecific antibody is an Fv unit,the variable domain of the heavy chain comprises a CDR1 having the aminoacid sequence of SEQ ID NO:25, a CDR2 having the amino acid sequence ofSEQ ID NO:26, and a CDR3 having the amino acid sequence of SEQ ID NO:27.In another embodiment, the heavy chain of the IL-17A/F binding entitycomprises a variable domain comprising the amino acid sequence of SEQ IDNO:13.

In another embodiment of the foregoing aspects of the invention, whenthe IL-23 binding entity of the bispecific antibody is an Fv unit, thevariable domain of the heavy chain comprises a CDR1 having the aminoacid sequence of SEQ ID NO:19, a CDR2 having the amino acid sequence ofSEQ ID NO:20, and a CDR3 having the amino acid sequence of SEQ ID NO:21.In another embodiment, the heavy chain of the IL-23 binding entitycomprises a variable domain comprising the amino acid sequence of SEQ IDNO:7.

In another embodiment of the foregoing aspects of the invention, the Fabfragments of the IL-23 binding entity of the bispecific antibody arelinked to the C-termini of the heavy chains (Fc) of the IL-17A/F bindingentity, or the Fab fragments of the IL-17A/F binding entity are linked,for example, to the C-termini of the heavy chains (Fc) of the IL-23binding entity by a linker molecule (see, for example, FIG. 2). Inanother embodiment, each of the light chain and the C_(H1) and variableregions of the heavy chain comprising the Fab fragments of the IL-23binding entity are linked to the N-termini of the light chain and heavychain, respectively, of the IL-17A/F binding entity, or each of thelight chain and the C_(H1) and variable regions of the heavy chaincomprising the Fab fragments of the IL-17A/F binding entity are linkedto the N-termini of the light chain and heavy chain, respectively, ofthe IL-23 binding entity by a linker molecule (see, for example, FIG.3). In another embodiment of the VCVFc bispecific antibody, each of thelight chain variable region and the heavy chain variable region of theFv unit comprising the second binding entity are linked to each of thelight chain constant region and the C_(H1) region, respectively, of theFab fragment of the first binding entity by a linker molecule (see FIG.5). Suitable linker molecules are known in the art and include, forexample, short polypeptides. A suitable linker may include a shortpolypeptide, which contains glycine, which confers flexibility, andserine or threonine, which confer solubility. A suitable linker maycomprise Gly₄Ser₁ units. For example, the linker may be (Gly₄Ser₁)_(x),wherein x is 1, 2, or 3. Optionally, the linker polypeptide has theamino acid sequence of SEQ ID NO:12. In another embodiment of the VCVFcbispecific antibodies, the linker for the light chain has the amino acidsequence of SEQ ID NO:85 and the linker for the heavy chain has theamino acid sequence of SEQ ID NO:86.

In another embodiment of the foregoing aspects of the invention, thebispecific antibody comprises a pair of heavy chains each comprising theamino acid sequence of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:74, or SEQ ID NO:84 and a pair of light chains eachcomprising the amino acid sequence of SEQ ID NO:17. In a preferredembodiment, the bispecific antibody comprises a pair of heavy chainscomprising the amino acid sequence of SEQ ID NO:74 and a pair of lightchains each comprising the amino acid sequence of SEQ ID NO:17.

In another embodiment of the foregoing aspects of the invention, anIL-17A/F antibody (or an antigen-binding fragment thereof) or theIL-17A/F binding entity of the bispecific antibody, such as a biAbFabL(see FIG. 2), a taFab (see FIG. 3), a heterodimeric Fc (see FIG. 4), aVCVFc (see FIG. 5) or a VCDFc (see FIG. 6) binds (a) an IL-17A homodimerwith a binding affinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹M, at least 1×10⁻¹⁰ M, at least 5×10⁻¹⁰ M, at least 8×10⁻¹⁰ M or atleast at least 1×10⁻¹¹ M; (b) an IL-17F homodimer with a bindingaffinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹ M, at least1×10⁻¹⁰ M, at least 2×10⁻¹⁰ M, at least 3×10⁻¹⁰ M, at least 4×10⁻¹⁰ M,at least 5×10⁻¹⁰ M or at least 1×10⁻¹¹ M; and/or (c) an IL-17A/Fheterodimer with a binding affinity (K_(D1)) of at least 1×10⁻⁸ M, atleast 5×10⁻⁸ M, at least 1×10⁻⁹ M, at least 2×10⁻⁹ M, at least 3×10⁻⁹ M,at least 4×10⁻⁹ M, at least 5×10⁻⁹ M, at least 6×10⁻⁹ M, at least 7×10⁻⁹M, at least 9×10⁻⁹ M, at least 1×10⁻¹⁰ M or at least 5×10⁻¹⁰ M, whereinthe binding affinity is measured by surface plasmon resonance, such asBiacore.

In another embodiment of the foregoing aspects of the invention, anIL-23p19 antibody (or an antigen-binding fragment thereof) or theIL-23p19 binding entity of the bispecific antibody, such as a biAbFabL(see FIG. 2), a taFab (see FIG. 3), a heterodimeric Fc (see FIG. 4), aVCVFc (see FIG. 5) or a VCDFc (see FIG. 6) binds IL-23p19 with a bindingaffinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹ M, at least1×10⁻¹⁰ M, at least 2×10⁻¹⁰ M, at least 3×10⁻¹⁰ M, at least 4×10⁻¹⁰ M,at least 5×10⁻¹⁰, at least 6×10⁻¹⁰, at least 7×10⁻¹⁰, at least 8×10⁻¹⁰or at least 9×10⁻¹⁰, at least 1×10⁻¹¹, wherein the binding affinity ismeasured by surface plasmon resonance, such as Biacore.

In another embodiment of the foregoing aspects of the invention, theIL-17A/F binding entity of the bispecific antibody, such as a biAbFabL(see FIG. 2), a taFab (see FIG. 3), a heterodimeric Fc (see FIG. 4), aVCVFc (see FIG. 5) or a VCDFc (see FIG. 6) binds (a) an IL-17A homodimerwith a binding affinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹M, at least 1×10⁻¹⁰ M, at least 5×10⁻¹⁰ M, at least 8×10⁻¹⁰ M or atleast at least 1×10⁻¹¹ M; (b) an IL-17F homodimer with a bindingaffinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹ M, at least1×10⁻¹⁰ M, at least 2×10⁻¹⁰ M, at least 3×10⁻¹⁰ M, at least 4×10⁻¹⁰ M,at least 5×10⁻¹⁰ M or at least 1×10⁻¹¹ M; and/or (c) an IL-17A/Fheterodimer with a binding affinity (K_(D1)) of at least 1×10⁻⁸ M, atleast 5×10⁻⁸ M, at least 1×10⁻⁹ M, at least 2×10⁻⁹ M, at least 3×10⁻⁹ M,at least 4×10⁻⁹ M, at least 5×10⁻⁹ M, at least 6×10⁻⁹ M, at least 7×10⁻⁹M, at least 9×10⁻⁹ M, at least 1×10⁻¹⁰ M or at least 5×10⁻¹⁰ M; and theIL-23p19 binding entity of the bispecific antibody binds IL-23p19 with abinding affinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹ M, atleast 1×10⁻¹⁰ M, at least 2×10⁻¹⁰ M, at least 3×10⁻¹⁰ M, at least4×10⁻¹⁰ M, at least 5×10⁻¹⁰, at least 6×10⁻¹⁰, at least 7×10⁻¹⁰, atleast 8×10⁻¹⁰ or at least 9×10⁻¹⁰, at least 1×10⁻¹¹, wherein the bindingaffinity is measured by surface plasmon resonance, such as Biacore.

In another embodiment of the foregoing aspects of the invention, anIL-17A/F antibody (or an antigen-binding fragment thereof) or theIL-17A/F binding entity of the bispecific antibody neutralizes orinhibits (a) IL-17A induction of G-CSF in primary human small airwayepithelial cells (SAEC) with an IC₅₀ of 0.5 μm or less; (b) IL-17Finduction G-CSF in primary human small airway epithelial cells (SAEC)with an IC₅₀ of 2.0 nM or less, 1.5 nM or less, 1.4 nM or less, 1.3 nMor less, 1.2 nM or less, 1.1 nM or less, or 1.0 nM or less; and/or (c)IL-17A/F induction G-CSF in primary human small airway epithelial cells(SAEC) with an IC₅₀ of 1.3 nM or less, 1.2 nM or less, 1.1 nM or less,1.0 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nMor less, or 0.5 nM or less.

In another embodiment of the foregoing aspects of the invention, anIL-17A/F antibody (or an antigen-binding fragment thereof) or theIL-17A/F binding entity of the bispecific antibody neutralizes orinhibits (a) IL-17A induction of IL-6 in human primary fibroblast cells(HFFF) with an IC₅₀ of 0.5 nM or less, 0.4 nM or less, 0.3 nM or less,0.2 nM or less, 0.1 nM or less, 0.09 nM or less, 0.08 nM or less, 0.07nM or less, 0.06 nM or less, 0.05 nM or less, 0.04 nM or less, 0.03 nMor less, 0.02 nM or less, or 0.01 nM or less; (b) IL-17F induction ofIL-6 in human primary fibroblast cells (HFFF) with an IC₅₀ of 30 nM orless, 28 nM or less, 26 nM or less, 25 nM or less, 22 nM or less, 20 nMor less, 19 nM or less, 18 nM or less, 17 nM or less, 16 nM or less, 15nM or less, 14 nM or less, 13 nM or less, 12 nM or less, 11 nM or less,or 10 nM or less; and/or (c) IL-17A/F induction of IL-6 in human primaryfibroblast cells (HFFF) with an IC₅₀ of 30 nM or less, 28 nM or less, 26nM or less, 22 nM or less, 20 nM or less, 18 nM or less, 17 nM or less,16 nM or less, 15 nM or less, 14 nM or less, 13 nM or less, 12 nM orless, 11 nM or less, 10 nM or less, 9.5 nM or less, 9.4 nM or less, 9.3nM or less, 9.2 nM or less, 9.1 nM or less, or 9.0 nM or less.

In another embodiment of the foregoing aspects of the invention, anIL-23p19 antibody (or an antigen-binding fragment thereof) or theIL-23p19 binding entity of the bispecific antibody neutralizes orinhibits (a) IL-23 induced IL-17A and IL-17F production in murinesplenocytes with an IC₅₀ of 0.5 nM or less, 0.4 nM or less, 0.3 nM orless, 0.2 nM or less, 0.1 nM or less, 0.09 nM or less, 0.08 nM or less,0.07 nM or less, or 0.06 nM or less.

In another embodiment of the foregoing aspects of the invention, anIL-23p19 antibody (or an antigen-binding fragment thereof) or theIL-23p19 binding entity of the bispecific antibody neutralizes orinhibits IL-23 induced STAT3 phosphorylation in activated primary humanT cells with an IC₅₀ of 0.1 nM or less, 0.2 nM or less, 0.3 nM or less,0.4 nM or less, 0.5 nM or less, 0.8 nM or less, 0.9 nM or less, 0.01 nMor less, 0.02 nM or less, 0.03 nM or less, 0.04 nM or less, or 0.05 nMor less.

In another embodiment of the foregoing aspects of the invention, theIL-17A/F binding entity of the bispecific antibody, such as a biAbFabL(see FIG. 2), a taFab (see FIG. 3), a heterodimeric Fc (see FIG. 4), aVCVFc (see FIG. 5) or a VCDFc (see FIG. 6) binds (a) an IL-17A homodimerwith a binding affinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹M, at least 1×10⁻¹⁰ M, at least 5×10⁻¹⁰ M, at least 8×10⁻¹⁰ M or atleast at least 1×10⁻¹¹ M; (b) an IL-17F homodimer with a bindingaffinity (K_(D1)) of at least 1×10⁻⁹ M, at least 5×10⁻⁹ M, at least1×10⁻¹⁰ M, at least 2×10⁻¹⁰ M, at least 3×10⁻¹⁰ M, at least 4×10⁻¹⁰ M,at least 5×10⁻¹⁰ M or at least 1×10⁻¹¹ M; and/or (c) an IL-17A/Fheterodimer with a binding affinity (K_(D1)) of at least 1×10⁻⁸ M, atleast 5×10⁻⁸ M, at least 1×10⁻⁹ M, at least 2×10⁻⁹ M, at least 3×10⁻⁹ M,at least 4×10⁻⁹ M, at least 5×10⁻⁹ M, at least 6×10⁻⁹ M, at least 7×10⁻⁹M, at least 9×10⁻⁹ M, at least 1×10⁻¹⁰ M or at least 5×10⁻¹⁰ M.Optionally, the IL-23p19 binding entity of the bispecific antibody bindsIL-23p19 with a binding affinity (K_(D1)) of at least 1×10⁻⁹ M, at least5×10⁻⁹ M, at least 1×10⁻¹⁰ M, at least 2×10⁻¹⁰ M, at least 3×10⁻¹⁰ M, atleast 4×10⁻¹⁰ M, at least 5×10⁻¹⁰, at least 6×10⁻¹⁰, at least 7×10⁻¹⁰,at least 8×10⁻¹⁰ or at least 9×10⁻¹⁰, at least 1×10⁻¹¹, wherein thebinding affinity is measured by surface plasmon resonance, such asBiacore. Optionally, the IL-17A/F binding entity of the bispecificantibody neutralizes or inhibits (a) IL-17A induction of G-CSF inprimary human small airway epithelial cells (SAEC) with an IC₅₀ of 0.5pm or less; (b) IL-17F induction G-CSF in primary human small airwayepithelial cells (SAEC) with an IC₅₀ of 2.0 nM or less, 1.5 nM or less,1.4 nM or less, 1.3 nM or less, 1.2 nM or less, 1.1 nM or less, or 1.0nM or less; and/or (c) IL-17A/F induction G-CSF in primary human smallairway epithelial cells (SAEC) with an IC₅₀ of 1.3 nM or less, 1.2 nM orless, 1.1 nM or less, 1.0 nM or less, 0.9 nM or less, 0.8 nM or less,0.7 nM or less, 0.6 nM or less, or 0.5 nM or less. Optionally, theIL-17A/F binding entity of the bispecific antibody neutralizes orinhibits (a) IL-17A induction of IL-6 in human primary fibroblast cells(HFFF) with an IC₅₀ of 0.5 nM or less, 0.4 nM or less, 0.3 nM or less,0.2 nM or less, 0.1 nM or less, 0.09 nM or less, 0.08 nM or less, 0.07nM or less, 0.06 nM or less, 0.05 nM or less, 0.04 nM or less, 0.03 nMor less, 0.02 nM or less, or 0.01 nM or less; (b) IL-17F induction ofIL-6 in human primary fibroblast cells (HFFF) with an IC₅₀ of 30 nM orless, 28 nM or less, 26 nM or less, 25 nM or less, 22 nM or less, 20 nMor less, 19 nM or less, 18 nM or less, 17 nM or less, 16 nM or less, 15nM or less, 14 nM or less, 13 nM or less, 12 nM or less, 11 nM or less,or 10 nM or less; and/or (c) IL-17A/F induction of IL-6 in human primaryfibroblast cells (HFFF) with an IC₅₀ of 30 nM or less, 28 nM or less, 26nM or less, 22 nM or less, 20 nM or less, 18 nM or less, 17 nM or less,16 nM or less, 15 nM or less, 14 nM or less, 13 nM or less, 12 nM orless, 11 nM or less, 10 nM or less, 9.5 nM or less, 9.4 nM or less, 9.3nM or less, 9.2 nM or less, 9.1 nM or less, or 9.0 nM or less.Optionally, the IL-23p19 binding entity of the bispecific antibodyneutralizes or inhibits IL-23 induced STAT3 phosphorylation in activatedprimary human T cells with an IC₅₀ of 0.1 nM or less, 0.2 nM or less,0.3 nM or less, 0.4 nM or less, 0.5 nM or less, 0.8 nM or less, 0.9 nMor less, 0.01 nM or less, 0.02 nM or less, 0.03 nM or less, 0.04 nM orless, or 0.05 nM or less.

In another embodiment of the foregoing aspects of the invention, thebispecific antibody comprises a pair of heavy chains comprising theamino acid sequence of SEQ ID NO:28 and a pair of light chainscomprising the amino acid sequence of SEQ ID NO:17 is referred to hereinas “biAb1”, “bAb1” or “23/17bAb1”. A bispecific antibody comprising apair of heavy chains each comprising the amino acid sequence of SEQ IDNO:18 and a pair of light chains each comprising the amino acid sequenceof SEQ ID NO:17 is referred to herein as “biAb2”, “bAb2” or “23/17bAb2”.A bispecific antibody comprising a pair of heavy chains each comprisingthe amino acid sequence of SEQ ID NO:74 and a pair of light chains eachcomprising the amino acid sequence of SEQ ID NO:17 is referred to hereinas “biAb3”, “bAb3” or “23/17bAb3”. A bispecific antibody comprising apair of heavy chains comprising the amino acid sequence of SEQ ID NO:29and a pair of light chains each comprising the amino acid sequence ofSEQ ID NO:17 is referred to herein as “biAb4”, “bAb4” or “23/17bAb4”.

In another embodiment of the foregoing aspects of the invention, thebispecific antibody comprises a pair of heavy chains each comprising theamino acid sequence of SEQ ID NO:77 and a pair of light chains eachcomprising the amino acid sequence of SEQ ID NO:79 and is referred toherein as “taFab1”.

In another embodiment of the foregoing aspects of the invention, thebispecific antibody comprises an IL-23 heavy chain comprising the aminoacid sequence of SEQ ID NO:63, an IL-17A/F heavy chain comprising theamino acid sequence of SEQ ID NO:65, and a pair of light chains eachcomprising the sequence of SEQ ID NO:17, and is referred to herein as“hetero1”. In another embodiment, the bispecific antibody comprises anIL-23 heavy chain comprising the amino acid sequence of SEQ ID NO:61, anIL-17A/F heavy chain comprising the amino acid sequence of SEQ ID NO:81,and a pair of light chains each comprising the sequence of SEQ ID NO:17,and is referred to herein as “hetero2”.

In another embodiment of the foregoing aspects of the invention, thebispecific antibody in the VCVFc format, see FIG. 5, has a pair of heavychains each comprising the amino acid sequence of SEQ ID NO:88 and apair of light chains each comprising the amino acid sequence of SEQ IDNO:90, or a pair of heavy chains each comprising the amino acid sequenceof SEQ ID NO:92 and a pair of light chains each comprising the aminoacid sequence of SEQ ID NO:94, or a pair of heavy chains each comprisingthe amino acid sequence of SEQ ID NO:96 and a pair of light chains eachcomprising the amino acid sequence of SEQ ID NO:90, or a pair of heavychains each comprising the amino acid sequence of SEQ ID NO:98 and apair of light chains each comprising the amino acid sequence of SEQ IDNO:94, or a pair of heavy chains each comprising the amino acid sequenceof SEQ ID NO:100 and a pair of light chains each comprising the aminoacid sequence of SEQ ID NO:102, or a pair of heavy chains eachcomprising the amino acid sequence of SEQ ID NO:104 and a pair of lightchains each comprising the amino acid sequence of SEQ ID NO:106, or apair of heavy chains each comprising the amino acid sequence of SEQ IDNO:112 and a pair of light chains each comprising the amino acidsequence of SEQ ID NO:114, or a pair of heavy chains each comprising theamino acid sequence of SEQ ID NO:116 and a pair of light chains eachcomprising the amino acid sequence of SEQ ID NO:118.

In another embodiment of the foregoing aspects of the invention, anisolated monoclonal antibody or antigen-binding fragment thereof thatspecifically binds to IL-17A (SEQ ID NO:2) and IL-17F (SEQ ID NO:4)comprising a heavy chain variable domain and a light chain variabledomain, wherein the heavy chain variable domain comprises the amino acidresidues of SEQ ID NO:13 and a light chain variable domain comprises theamino acid residues of SEQ ID NO:9. Optionally, the monoclonal antibodycomprises a human constant region, e.g., IgG1, IgG2, IgG3 or IgG4. TheIgG4 human constant region may have a Serine to Proline mutation atposition 241 according to Kabat. Optionally, the heavy chain comprisesthe amino acid residues of SEQ ID NOs:16, 18, 28, 29 or 74. Optionally,the light chain comprises the amino acid residues of SEQ ID NO:17.Optionally, the heavy chain comprises the amino acid residues of SEQ IDNOs:16, 18, 28, 29 or 74, and the light chain comprises the amino acidresidues of SEQ ID NO:17. Optionally, a bispecific antibody comprisesthe monoclonal antibody.

Heavy chain and light chain constant regions include, IgG1.1 (SEQ IDNO:11, which may be encoded by SEQ ID NO:82), IgG1.1f without aC-terminal Lysine (SEQ ID NO:127), IgG1.1f with a C-terminal Lysine (SEQID NO:128), human kappa constant region (SEQ ID NO:10, which may beencoded by SEQ ID NO:83), or IgG4.1 (SEQ ID NO:8). The IgG4 heavy chainconstant domain may include a variant of wild-type IgG4 that has amutation in the hinge region, S228P (EU index numbering system) or S241P(Kabat numbering system). Changing the serine at 241 (Kabat) to proline(found at that position in IgG1 and IgG2) in a mouse/human chimericheavy chain leads to the production of a homogeneous antibody andabolishes the heterogeneity. Further, the variant IgG4 has significantlyextended serum half-life and shows an improved tissue distributioncompared to the original chimeric IgG4. Angal et al., MolecularImmunology, 30(1):105-108 (1993); Schuurman et al., MolecularImmunology, 38:1-8 (2001); Lewis et al., Molecular Immunology,46:3488-3494 (2009).

In another embodiment of the foregoing aspects of the invention, anisolated monoclonal antibody or antigen-binding fragment thereof thatspecifically binds to IL-23p19 (SEQ ID NO:6) comprising a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises the amino acid residues of SEQ ID NO:7,and wherein the light chain variable domain comprises the amino acidresidues of SEQ ID NO:9. Optionally, the monoclonal antibody comprises ahuman constant region, e.g., IgG1, IgG2, IgG3 or IgG4. Optionally, theIgG4 human constant region has a Serine to Proline mutation at position241 according to Kabat. Optionally, the heavy chain comprises the aminoacid residues of SEQ ID NOs:16, 18, 28, 29 or 74. Optionally, the lightchain comprises the amino acid residues of SEQ ID NO:17. Optionally, theheavy chain comprises the amino acid residues of SEQ ID NOs:16, 18, 28,29 or 74, and the light chain comprises the amino acid residues of SEQID NO:17. Optionally, a bispecific antibody comprises the monoclonalantibody.

In another embodiment of the foregoing aspects of the invention, theantibody, bispecific antibody, or antigen-binding fragment thereof,specifically binds IL-23p19, wherein the antibody or antigen-bindingfragment binds a discontinuous epitope on IL-23p19 comprising a firstepitope and a second epitope, wherein the first epitope consists of atleast one amino acid of amino acid residues 33-59 of SEQ ID NO:6 and thesecond epitope consists of at least one amino acid of amino acidresidues 89-125 of SEQ ID NO:6. Optionally, the antibody, bispecificantibody, or antigen-binding fragment thereof binds to at least aminoacid residue 54 of SEQ ID NO:6 of the first epitope. Optionally, theantibody, bispecific antibody, or antigen-binding fragment thereof bindsto at least amino acid residue 55 of SEQ ID NO:6 of the first epitope.Optionally, the antibody, bispecific antibody, or antigen-bindingfragment thereof binds to at least amino acid residues 54 and 55 of SEQID NO:6 of the first epitope. Optionally, the antibody, bispecificantibody, or antigen-binding fragment thereof binds to at least aminoacid residue 116 of SEQ ID NO:6 of the second epitope. Optionally, theantibody, bispecific antibody, or antigen-binding fragment thereof bindsto at least amino acid residues 54 and 55 of SEQ ID NO:6 of the firstepitope, and to least amino acid residue 116 of SEQ ID NO:6 of thesecond epitope.

In another embodiment of the foregoing aspects of the invention, theantibody, bispecific antibody, or antigen-binding fragment thereofspecifically binds IL-23p19, wherein the antibody or antigen-bindingfragment binds a discontinuous epitope on IL-23p19 comprising a firstepitope and a second epitope, wherein the antibody or antigen-bindingfragment binds to at least amino acid residues 54 and 55 of SEQ ID NO:6of the first epitope, and to least amino acid residue 116 of SEQ ID NO:6of the second epitope.

In another embodiment of the foregoing aspects of the invention, anIL-17A/F binding entity specifically binds IL-17A (IL-17A/IL-17Ahomodimer and IL-17A/IL-17F heterodimer) at an epitope comprising atleast amino acid residue 108 (Tyr) of SEQ ID NO:2, wherein the IL-17A/Fbinding entity is a monoclonal antibody or antigen-binding fragmentthereof. Optionally, the epitope on IL-17A is determined by alaninemutagenesis and/or X-ray crystallography. The epitope on which theIL-17A/F binding entity binds IL-17A may be a continuous or adiscontinuous epitope.

In another embodiment of the foregoing aspects of the invention, theIL-17A/F cross-reactive monoclonal antibody or an tigen-binding fragmentthereof binds IL-17A at an epitope comprising at least amino acidresidue 108 (Tyr) of SEQ ID NO:2. Optionally, the epitope on IL-17A isdetermined by alanine mutagenesis and/or X-ray crystallography. Theepitope on which the IL-17A/F cross-reactive monoclonal antibody or antigen-binding fragment thereof binds IL-17A may be a continuous or adiscontinuous epitope.

The bispecific antibodies of the invention may be used alone or asimmunoconjugates with a cytotoxic agent. In some embodiments, the agentis a chemotherapeutic agent. In some embodiments, the agent is aradioisotope, including, but not limited to Lead-212, Bismuth-212,Astatine-211, Iodine-131, Scandium-47, Rhenium-186, Rhenium-188,Yttrium-90, Iodine-123, Iodine-125, Bromine-77, Indium-111, andfissionable nuclides such as Boron-10 or an Actinide. In otherembodiments, the agent is a toxin or cytotoxic drug, including but notlimited to ricin, abrin, modified Pseudomonas enterotoxin A, Pseudomonasexotoxin, calicheamicin, adriamycin, 5-fluorouracil, diphtheria toxin,and the like. Methods of conjugation of antibodies to such agents areknown in the literature, and include direct and indirect conjugation.

Suitable detectable molecules may be directly or indirectly attached tothe antibodies of the present invention. Suitable detectable moleculesinclude radionuclides, enzymes, substrates, cofactors, inhibitors,fluorescent markers, chemiluminescent markers, magnetic particles andthe like. For indirect attachment of a detectable or cytotoxic molecule,the detectable or cytotoxic molecule can be conjugated with a member ofa complementary/anticomplementary pair, where the other member is boundto the binding polypeptide or antibody portion. For these purposes,biotin/streptavidin is an exemplary complementary/anticomplementarypair.

The bispecific antibodies, antibodies and antigen-binding fragments ofthe invention also include derivatives that are modified, e.g., by thecovalent attachment of any type of molecule to the antibody such thatcovalent attachment does not prevent the antibody from binding to itsepitope. Examples of suitable derivatives include, but are not limitedto fucosylated antibodies, glycosylated antibodies, acetylatedantibodies, pegylated antibodies, phosphorylated antibodies, andamidated antibodies. The antibodies and derivatives thereof of theinvention may themselves by derivatized by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherproteins, and the like. In some embodiments of the invention, at leastone heavy chain of the antibody is fucosylated. In some embodiments, thefucosylation is N-linked. In some preferred embodiments, at least oneheavy chain of the antibody comprises a fucosylated, N-linkedoligosaccharide.

The bispecific antibodies, antibodies and antigen-binding fragments ofthe invention include variants having single or multiple amino acidsubstitutions, deletions, additions, or replacements that retain thebiological properties (e.g., block the binding of IL-17A or IL-17Fand/or IL-23 to their respective receptors, inhibit the biologicalactivity of IL-17A or IL-17F and IL-23) of the antibodies of theinvention. The skilled person can produce variants having single ormultiple amino acid substitutions, deletions, additions or replacements.These variants may include, inter alia: (a) variants in which one ormore amino acid residues are substituted with conservative ornonconservative amino acids, (b) variants in which one or more aminoacids are added to or deleted from the polypeptide, (c) variants inwhich one or more amino acids include a substituent group, and (d)variants in which the polypeptide is fused with another peptide orpolypeptide such as a fusion partner, a protein tag or other chemicalmoiety, that may confer useful properties to the polypeptide, such as,for example, an epitope for an antibody, a polyhistidine sequence, abiotin moiety and the like. Antibodies of the invention may includevariants in which amino acid residues from one species are substitutedfor the corresponding residue in another species, either at theconserved or nonconserved positions. In another embodiment, amino acidresidues at nonconserved positions are substituted with conservative ornonconservative residues. The techniques for obtaining these variants,including genetic (suppressions, deletions, mutations, etc.), chemical,and enzymatic techniques, are known to the person having ordinary skillin the art.

The invention also includes isolated nucleic acids encoding thebispecific antibodies of the invention, which includes, for instance,the light chain, light chain variable region, light chain constantregion, heavy chain, heavy chain variable region, heavy chain constantregion, linkers, and any and all components and combinations thereof ofthe bispecific antibodies disclosed herein. Nucleic acids of theinvention include nucleic acids having at least 80%, more preferably atleast about 90%, more preferably at least about 95%, and most preferablyat least about 98% homology to nucleic acids of the invention. The terms“percent similarity”, “percent identity” and “percent homology” whenreferring to a particular sequence are used as set forth in theUniversity of Wisconsin GCG® software program. Nucleic acids of theinvention also include complementary nucleic acids. In some instances,the sequences will be fully complementary (no mismatches) when aligned.In other instances, there may be up to about a 20% mismatch in thesequences. In some embodiments of the invention are provided nucleicacids encoding both a heavy chain and a light chain of an antibody ofthe invention.

Nucleic acids of the invention can be cloned into a vector, such as aplasmid, cosmid, bacmid, phage, artificial chromosome (BAC, YAC) orvirus, into which another genetic sequence or element (either DNA orRNA) may be inserted so as to bring about the replication of theattached sequence or element. In some embodiments, the expression vectorcontains a constitutively active promoter segment (such as but notlimited to CMV, SV40, Elongation Factor or LTR sequences) or aninducible promoter sequence such as the steroid inducible pIND vector(Invitrogen), where the expression of the nucleic acid can be regulated.Expression vectors of the invention may further comprise regulatorysequences, for example, an internal ribosomal entry site. The expressionvector can be introduced into a cell by transfection, for example.

Thus in another embodiment, the present invention provides an expressionvector comprising the following operably linked elements; atranscription promoter; a nucleic acid molecule encoding the heavy chainof a bispecific antibody of the invention; and a transcriptionterminator. In another embodiment, the present invention provides anexpression vector comprising the following operably linked elements; atranscription promoter; a nucleic acid molecule encoding the light chainof a bispecific antibody of the invention; and a transcriptionterminator. Recombinant host cells comprising such vectors andexpressing the heavy and light chains are also provided.

In another embodiment, the present invention provides an expressionvector comprising the following operably linked elements; atranscription promoter; a first nucleic acid molecule encoding the heavychain of a bispecific antibody, antibody or antigen-binding fragment ofthe invention; a second nucleic acid molecule encoding the light chainof a bispecific antibody, antibody or antigen-binding fragment of theinvention; and a transcription terminator. In another embodiment, thepresent invention provides an expression vector comprising the followingoperably linked elements; a first transcription promoter; a firstnucleic acid molecule encoding the heavy chain of a bispecific antibody,antibody or antigen-binding fragment of the invention; a firsttranscription terminator; a second transcription promoter a secondnucleic acid molecule encoding the light chain of a bispecific antibody,antibody or antigen-binding fragment of the invention; and a secondtranscription terminator. Recombinant host cells comprising such vectorsand expressing the heavy and light chains are also provided.

Antibody-producing cells containing a nucleic acid encoding the heavychain and a nucleic acid encoding the light chain of the bispecificantibodies, antibodies or antigen-binding fragments of the invention canbe used to produce the bispecific antibodies, antibodies orantigen-binding fragments in accordance with techniques known in theart. The present invention, in one embodiment, provides a method ofproducing a bispecific antibody, antibody or antigen-binding fragment ofthe invention comprising culturing a recombinant host cell expressingthe heavy and light chains and isolating the bispecific antibody,antibody or antigen-binding fragment produced by the cell.

The recombinant host cell may be a prokaryotic cell, for example a E.coli cell, or a eukaryotic cell, for example a mammalian cell or a yeastcell. Yeast cells include Saccharomyces cerevisiae, Schizosaccharomycespombe, and Pichia pastoris cells. Mammalian cells include VERO, HeLa,Chinese hamster Ovary (CHO), W138, baby hamster kidney (BHK), COS-7,MDCK, human embryonic kidney line 293, normal dog kidney cell lines,normal cat kidney cell lines, monkey kidney cells, African green monkeykidney cells, COS cells, and non-tumorigenic mouse myoblast G8 cells,fibroblast cell lines, myeloma cell lines, mouse NIH/3T3 cells, LMTK31cells, mouse sertoli cells, human cervical carcinoma cells, buffalo ratliver cells, human lung cells, human liver cells, mouse mammary tumorcells, TRI cells, MRC 5 cells, and FS4 cells. Antibody-producing cellsof the invention also include any insect expression cell line known,such as for example, Spodoptera frugiperda cells. In a preferredembodiment, the cells are mammalian cells. In another preferredembodiment, the mammalian cells are CHO cells.

The antibody-producing cells preferably are substantially free ofIL-17A, IL-17F and IL-23 binding competitors. In preferred embodiments,the antibody-producing cells comprise less than about 10%, preferablyless than about 5%, more preferably less than about 1%, more preferablyless than about 0.5%, more preferably less than about 0.1%, and mostpreferably 0% by weight IL-17A, IL-17F, or IL-23 binding competitors. Insome embodiments, the antibodies produced by the antibody-producingcells are substantially free of IL-17A, IL-17F, and IL-23 competitors.In preferred embodiments, antibodies produced by the antibody-producingcells comprise less than about 10%, preferably less than about 5%, morepreferably less than about 1%, more preferably less than about 0.5%,more preferably less than about 0.1%, and most preferably 0% by weightboth IL-17 and IL-23 binding competitors.

Methods of antibody purification are known in the art. In someembodiments of the invention, methods for antibody purification includefiltration, affinity column chromatography, cation exchangechromatography, anion exchange chromatography, and concentration. Thefiltration step preferably comprises ultrafiltration, and morepreferably ultrafiltration and diafiltration. Filtration is preferablyperformed at least about 5-50 times, more preferably 10 to 30 times, andmost preferably 14 to 27 times. Affinity column chromatography, may beperformed using, for example, PROSEP® Affinity Chromatography(Millipore, Billerica, Mass.). In a preferred embodiment, the affinitychromatography step comprises PROSEP®-vA column chromatography. Eluatemay be washed in a solvent detergent. Cation exchange chromatography mayinclude, for example, SP-Sepharose Cation Exchange Chromatography. Anionexchange chromatography may include, for example but not limited to,Q-Sepharose Fast Flow Anion Exchange. The anion exchange step ispreferably non-binding, thereby allowing removal of contaminantsincluding DNA and BSA. The antibody product is preferably nanofiltered,for example, using a Pall DV 20 Nanofilter. The antibody product may beconcentrated, for example, using ultrafiltration and diafiltration. Themethod may further comprise a step of size exclusion chromatography toremove aggregates.

The bispecific antibodies, antibodies or antigen-binding fragments mayalso be produced by other methods known in the art, for example bychemical coupling of antibodies and antibody fragments.

The bispecific antibodies, antibodies or antigen-binding fragments ofthe present invention are useful, for example, for the inhibition ofproinflammatory cytokines, such as IL-17A, IL-17F and IL-23/p19. Theantibodies can be used to reduce, limit, neutralize, or block theproinflammatory effects of the IL-17A homodimer, the IL-17F homodimer,and/or the IL-17A/F heterodimer. Likewise, the antibodies can be used toreduce, limit, neutralize, or block the pro-cancerous effects of theIL-17A homodimer, the IL-17F homodimer, or the IL-17A/F heterodimer. Insuch cases, the anti-IL-23p19 portion of the antibody is used to reduce,limit, neutralize, or block production of new T cells that would produceIL-17A and/or IL-17F, including homodimers and heterodimers. Thebispecific antibodies, antibodies or antigen-binding fragments describedherein can be used to treat inflammatory disorders and autoimmunediseases, such as multiple sclerosis (e.g., relapsing-remitting multiplesclerosis, secondary-progressive multiple sclerosis, primary-progressivemultiple sclerosis and progressive-relapsing multiple sclerosis), cysticfibrosis, inflammatory bowel disease, psoriasis, systemic sclerosis,systemic lupus erythematosus, lupus nephritis, IgA nephropathy, diabetickidney disease, minimal change disease (lipoid nephrosis), focalsegmental glomerulosclerosis (FSGS), nephrogenic systemic fibrosis(NSF), nephrogenic fibrosing dermopathy, fibrosing cholestatichepatitis, eosinophilic fasciitis (Shulman's syndrome), scleromyxedema(popular mucinosis), scleroderma, lichen sclerosusetatrophicus, POEMssyndrome (Crow-Fukase syndrome, Takatsuki disease or PEP syndrome),nephrotic syndrome, graft-versus-host-disease (GVHD),graft-versus-host-disease (GVHD) (from a transplant, such as blood, bonemarrow, kidney, pancreas, liver, orthotopic liver, lung, heart,intestine, small intestine, large intestine, thymus, allogeneic stemcell, reduced-intensity allogeneic, bone, tendon, cornea, skin, heartvalves, veins, arteries, blood vessels, stomach and testis),antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis(AAV), giant cell arteritis and multiple-myeloma-induced lytic bonedisease. The bispecific antibodies, antibodies or antigen-bindingfragments described herein can also be used to treat cancer, includingangiogenesis.

The bispecific antibodies, antibodies or antigen-binding fragments ofthe present invention inhibit the activity of IL-17A and/or IL-17F andIL-23 (via the p19 subunit), and thus, inhibit the production,maintenance, and activity of new and existing IL-17A and IL-17F andIL-17-producing T cells (Th17). The invention further concerns the useof the bispecific antibodies, antibodies or antigen-binding fragments ofthe present invention in the treatment of inflammatory diseasescharacterized by the presence of elevated levels of IL-17A, IL-17F,and/or IL-23, and in the treatment of cancers characterized by thepresence of elevated levels of IL-17A, IL-17F, and/or IL-23.

The bispecific antibodies, antibodies or antigen-binding fragments ofthe present invention may block, inhibit, reduce, antagonize orneutralize the activity of IL-17A, IL-17F, (including both homodimersand the heterodimer), and IL-23/p19 thus are advantageous over therapiesthat target only one or two of these three cytokines.

The antibodies, e.g., bispecific antibodies, of the invention are thususeful to:

(1) Block, inhibit, reduce, antagonize or neutralize signaling viaIL-17A or IL-17F and IL-23 in the treatment of cancer, acuteinflammation, and chronic inflammatory diseases such as inflammatorybowel disease (IBD), Crohn's disease, ulcerative colitis, irritablebowel syndrome (IBS), cystic fibrosis, chronic colitis, Sjögren'ssyndrome, splenomegaly, inflammation in chronic kidney disease (CKD),psoriasis, psoriatic arthritis, rheumatoid arthritis, and other diseasesassociated with the induction of acute-phase response.

(2) Block, inhibit, reduce, antagonize or neutralize signaling viaIL-17A or IL-17F or IL-23 in the treatment of autoimmune diseases suchas insulin-dependent diabetes mellitus (IDDM), multiple sclerosis (MS)(e.g., relapsing-remitting multiple sclerosis, secondary-progressivemultiple sclerosis, primary-progressive multiple sclerosis andprogressive-relapsing multiple sclerosis), systemic Lupus erythematosus(SLE), myasthenia gravis, rheumatoid arthritis, Sjögren's syndrome, IBSand IBD to prevent or inhibit signaling in immune cells (e.g.,lymphocytes, monocytes, leukocytes) via their receptors (e.g., IL-23Rα,IL-12Rβ1, IL-17RA and IL-17RC). Blocking, inhibiting, reducing, orantagonizing signaling via IL-23Rα, IL-12Rβ1, IL-17RA and IL-17RC, usingthe antibodies of the present invention, also benefits diseases of thepancreas, kidney, pituitary and neuronal cells and may be used to treatIDDM, non-insulin dependent diabetes mellitus (NIDDM), pancreatitis, andpancreatic carcinoma.

For example, the bispecific antibodies, antibodies or antigen-bindingfragments of the present invention are useful in therapeutic treatmentof inflammatory diseases, particularly as antagonists to IL-17A, IL-17F,and IL-23/p19, in the treatment of inflammatory diseases such asmultiple sclerosis (MS) (e.g., relapsing-remitting multiple sclerosis,secondary-progressive multiple sclerosis, primary-progressive multiplesclerosis and progressive-relapsing multiple sclerosis), inflammatorybowel disease (IBD), and cancer. These antagonists are capable ofbinding, blocking, inhibiting, reducing, antagonizing or neutralizingIL-17A, IL-17F, their homodimers and heterodimers, and IL-23 (via p19)(either individually or together) in the treatment of atopic and contactdermatitis, systemic sclerosis, systemic lupus erythematosus (SLE),antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis(AAV), giant cell arteritis, multiple sclerosis (MS) (e.g.,relapsing-remitting multiple sclerosis, secondary-progressive multiplesclerosis, primary-progressive multiple sclerosis andprogressive-relapsing multiple sclerosis), colitis, endotoxemia,arthritis, rheumatoid arthritis (RA), Sjögren's syndrome, psoriaticarthritis, adult respiratory disease (ARD), septic shock, multiple organfailure, inflammatory lung injury such as idiopathic pulmonary fibrosis,asthma, chronic obstructive pulmonary disease (COPD), airwayhyper-responsiveness, chronic bronchitis, allergic asthma, psoriasis,eczema, IBS and inflammatory bowel disease (IBD) such as ulcerativecolitis and Crohn's disease, Helicobacter pylori infection, lupusnephritis, IgA nephropathy, diabetic kidney disease, minimal changedisease (lipoid nephrosis), focal segmental glomerulosclerosis (FSGS),nephrogenic systemic fibrosis (NSF), nephrogenic fibrosing dermopathy,fibrosing cholestatic hepatitis, eosinophilic fasciitis (Shulman'ssyndrome), scleromyxedema (popular mucinosis), scleroderma, lichensclerosusetatrophicus, POEMs syndrome (Crow-Fukase syndrome, Takatsukidisease or PEP syndrome), nephrotic syndrome, transplant rejection,graft-versus-host-disease (GVHD), graft-versus-host-disease (GVHD) (froma transplant, such as blood, bone marrow, kidney, pancreas, liver,orthotopic liver, lung, heart, intestine, small intestine, largeintestine, thymus, allogeneic stem cell, reduced-intensity allogeneic,bone, tendon, cornea, skin, heart valves, veins, arteries, bloodvessels, stomach and testis), intraabdominal adhesions and/or abscessesas results of peritoneal inflammation (e.g., from infection, injury,etc.), nephrotic syndrome, cystic fibrosis (Tan, H.-L. et al., AmericanJournal of Respiratory and Critical Care Medicine, 184(2):252-258(2011)), lytic bone disease (e.g., multiple-myeloma-induced lytic bonedisease) (Sotomayor, E. M., Blood, 116(18):3380-3382 (2010)), organallograft rejection, streptococcal cell wall (SCW)-induced arthritis,osteoarthritis, gingivitis/periodontitis, herpetic stromal keratitis,restenosis, Kawasaki disease, age-related macular degeneration (AMD;e.g., wet form of AMD and dry form of AMD) (Wei, L. et al., CellReports, 2:1151-1158 (Nov. 29, 2012), immune mediated renal diseases,liver fibrosis (Meng, F. et al., Gastroenterology, 143:765-776 (2012),pulmonary fibrosis (Meng, F. et al., Gastroenterology, 143:765-776(2012), hepatobiliary diseases, myocarditis (Ding, H.-S., Mol. Biol.Rep., 39(7):7473-7478 (Feb. 14, 2012); Valente, A. J. et al., CellularSignalling, 24:560-568 (2012)), cardiac fibrosis (Valente, A. J. et al.,Cellular Signalling, 24:560-568 (2012)), adverse myocardial remodeling(Valente, A. J. et al., Cellular Signalling, 24:560-568 (2012)),atherosclerosis (Ding, H.-S., Mol. Biol. Rep., 39(7):7473-7478 (Feb. 14,2012), cardiac ischemia/reperfusion injury (Ding, H.-S., Mol. Biol.Rep., 39(7):7473-7478 (Feb. 14, 2012), heart failure (Ding, H.-S., Mol.Biol. Rep., 39(7):7473-7478 (Feb. 14, 2012) and cancers/neoplasticdiseases that are characterized by IL-17 and/or IL-23 expression,including but not limited to prostate, renal, colon, ovarian andcervical cancer, and leukemias (Tartour et al., Cancer Res., 59:3698(1999); Kato et al., Biochem. Biophys. Res. Commun., 282:735 (2001);Steiner et al., Prostate, 56:171 (2003); Langowksi et al., Nature, May10 [Epub ahead of print], (2006)).

For example, the bispecific antibodies, antibodies or antigen-bindingfragments of the present invention are useful, e.g., antagonists toIL-17A, IL-17F, and IL-23/p19, in therapeutic treatment of inflammatorydiseases, particularly in the treatment of Acquired ImmunodeficiencySyndrome (AIDS, which is a viral disease with an autoimmune component),alopecia areata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmunehepatitis, autoimmune inner ear disease (AIED), autoimmunelymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura(ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitishepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS),chronic inflammatory demyelinating polyneuropathy (CIPD), cicatricialpemphigold, cold agglutinin disease, crest syndrome, Degos' disease,dermatomyositis-juvenile, discoid lupus (e.g., childhood discoid lupuserythematosus, generalized discoid lupus erythematosus and localizeddiscoid lupus erythematosus), chilblain lupus erythematosus, lupuserythematosus-lichen planus overlap syndrome, lupus erythematosuspanniculitis, tumid lupus erythematosus, verrucous lupus erythematosuscutaneous, systemic lupus erythematosus, subacute cutaneous lupuserythematosus, acute cutaneous lupus erythematosus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease,Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonaryfibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy,insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still'sdisease), juvenile rheumatoid arthritis, rheumatoid arthritis (RA),Meniere's disease, mixed connective tissue disease, multiple sclerosis(MS) (e.g., relapsing-remitting multiple sclerosis,secondary-progressive multiple sclerosis, primary-progressive multiplesclerosis and progressive-relapsing multiple sclerosis), myastheniagravis, pernacious anemia, polyarteritis nodosa, polychondritis,polyglandular syndromes, polymyalgia rheumatica, polymyositis anddermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis,eczema, psoriasis, psoriatic arthritis, Raynaud's phenomena, Reiter'ssyndrome, adult respiratory disease (ARD), rheumatic fever, arthritis,sarcoidosis, scleroderma (e.g., progressive systemic sclerosis (PSS),also known as systemic sclerosis (SS)), Sjögren's syndrome, stiff-mansyndrome, systemic lupus erythematosus (SLE), antineutrophil cytoplasmicantibodies (ANCA)-associated vasculitis (AAV), giant cell arteritis,Takayasu arteritis, temporal arteritis/giant cell arteritis, endotoxia,sepsis or septic shock, toxic shock syndrome, multiple organ failure,inflammatory lung injury such as idiopathic pulmonary fibrosis, colitis,inflammatory bowel disease (IBD) such as ulcerative colitis and Crohn'sdisease, irritable bowel syndrome (IBS), uveitis, vitiligo, Wegener'sgranulomatosis, Alzheimer's disease, atopic allergy, allergy, asthma,bronchial asthma, chronic obstructive pulmonary disease (COPD), airwayhyper-responsiveness, allergic asthma, glomerulonephritis, hemolyticanemias, Helicobacter pylori infection, intraabdominal adhesions and/orabscesses as results of peritoneal inflammation (e.g., from infection,injury, etc.), nephrotic syndrome, idiopathic demyelinatingpolyneuropathy, Guillain-Barre syndrome, organ allograft rejection,lupus nephritis, IgA nephropathy, diabetic kidney disease, minimalchange disease (lipoid nephrosis), focal segmental glomerulosclerosis(FSGS), nephrogenic systemic fibrosis (NSF), nephrogenic fibrosingdermopathy, fibrosing cholestatic hepatitis, eosinophilic fasciitis(Shulman's syndrome), scleromyxedema (popular mucinosis), scleroderma,lichen sclerosusetatrophicus, POEMs syndrome (Crow-Fukase syndrome,Takatsuki disease or PEP syndrome), nephrotic syndrome,graft-versus-host-disease (GVHD), graft-versus-host-disease (GVHD) (froma transplant, such as blood, bone marrow, kidney, pancreas, liver,orthotopic liver, lung, heart, intestine, small intestine, largeintestine, thymus, allogeneic stem cell, reduced-intensity allogeneic,bone, tendon, cornea, skin, heart valves, veins, arteries, bloodvessels, stomach and testis), lytic bone disease (e.g., multiplemyeloma-induced lytice bone disease), cystic fibrosis, age-relatedmascular degeneration (AMD; e.g., wet AMD and dry AMD), liver fibrosis,pulmonary fibrosis, atherosclerosis, cardiac ischemia/reperfusioninjury, heart failure, myocarditis, cardiac fibrosis, adverse myocardialremodeling, diabetic retinopathy and ventilator induced lung injury.

Accordingly, in one embodiment, the present invention provides a methodof inhibiting one or more of proinflammatory cytokines, e.g., IL-17A,IL-17F and IL-23, in a mammal in need of such treatment comprisingadministering a therapeutically effective amount of a bispecificantibody, antibody or antigen-binding fragment to a mammal in need ofsuch treatment. In a preferred embodiment, the mammal is a human. Themethod may be used to treat a disorder characterized by elevatedexpression of IL-17A, IL-17F, or IL-23. The bispecific antibody,antibody or antigen-binding fragment maybe administered with anotherpharmaceutical agent, either in the same formulation or separately.

In another embodiment, the present invention provides a method oftreating an immune related disorder in a mammal in need thereof,comprising administering to the mammal a therapeutically effectiveamount of an IL-17A/F polypeptide, an agonist thereof, or an antagonist(such as an IL-17A/F binding entity which includes an IL-17A/Fcross-reactive antibody) thereto. In a preferred aspect, the immunerelated disorder is selected form the group consisting of: systemiclupus erythematosis (SLE), rheumatoid arthritis (RA), osteoarthritis,juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis,idiopathic inflammatory myopathies, Sjögren's syndrome, systemicvasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmunethrombocytopenia, thyroiditis, diabetes mellitus, immune-mediated renaldisease, demyelinating diseases of the central and peripheral nervoussystems such as multiple sclerosis (MS) (e.g., relapsing-remittingmultiple sclerosis, secondary-progressive multiple sclerosis,primary-progressive multiple sclerosis and progressive-relapsingmultiple sclerosis), idiopathic demyelinating polyneuropathy orGuillain-Barre syndrome, and chronic inflammatory demyelinatingpolyneuropathy, hepatobiliary diseases such as infectious, autoimmunechronic active hepatitis, primary biliary cirrhosis, granulomatoushepatitis, and sclerosing cholangitis, inflammatory bowel disease (IBD),Crohn's disease, ulcerative colitis, gluten-sensitive enteropathy, andWhipple's disease, autoimmune or immune-mediated skin diseases includingbullous skin diseases, erythema multiforme and contact dermatitis,antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis(AAV), giant cell arteritis, psoriasis, psoriatic arthritis, allergicdiseases such as asthma, allergic rhinitis, atopic dermatitis, foodhypersensitivity and urticaria, immunologic diseases of the lung such aseosinophilic pneumonia, idiopathic pulmonary fibrosis andhypersensitivity pneumonitis, lupus nephritis, IgA nephropathy, diabetickidney disease, minimal change disease (lipoid nephrosis), focalsegmental glomerulosclerosis (FSGS), nephrogenic systemic fibrosis(NSF), nephrogenic fibrosing dermopathy, fibrosing cholestatichepatitis, eosinophilic fasciitis (Shulman's syndrome), scleromyxedema(popular mucinosis), scleroderma, lichen sclerosusetatrophicus, POEMssyndrome (Crow-Fukase syndrome, Takatsuki disease or PEP syndrome),nephrotic syndrome, graft-versus-host-disease (GVHD),graft-versus-host-disease (GVHD) (from a transplant, such as blood, bonemarrow, kidney, pancreas, liver, orthotopic liver, lung, heart,intestine, small intestine, large intestine, thymus, allogeneic stemcell, reduced-intensity allogeneic, bone, tendon, cornea, skin, heartvalves, veins, arteries, blood vessels, stomach and testis), lytic bonedisease (e.g., multiple myeloma-induced lytice bone disease), cysticfibrosis, age-related mascular degeneration (AMD; e.g., wet AMD and dryAMD), liver fibrosis, pulmonary fibrosis, atherosclerosis, cardiacischemia/reperfusion injury, heart failure, myocarditis, cardiacfibrosis, adverse myocardial remodeling, transplantation associateddiseases including graft rejection and graft-versus-host-disease.

In another embodiment, the present invention provides a method forinhibiting inflammation in a mammal in need of such treatment comprisingadministering a therapeutically effective amount of a bispecificantibody, antibody or antigen-binding fragment of the invention to amammal in need of such treatment. In a preferred embodiment, the mammalis a human. The inflammation may be associated with a disease selectedfrom the group consisting of multiple sclerosis (MS) (e.g.,relapsing-remitting multiple sclerosis, secondary-progressive multiplesclerosis, primary-progressive multiple sclerosis andprogressive-relapsing multiple sclerosis), chronic inflammation,Sjögren's syndrome, autoimmune diabetes, rheumatoid arthritis (RA) andother arthritic conditions, asthma, systemic sclerosis, atopicdermatitis, antineutrophil cytoplasmic antibodies (ANCA)-associatedvasculitis (AAV), giant cell arteritis, systemic lupus erythematosus(SLE), Degos' disease, dermatomyositis-juvenile, discoid lupus (e.g.,childhood discoid lupus erythematosus, generalized discoid lupuserythematosus and localized discoid lupus erythematosus), chilblainlupus erythematosus, lupus erythematosus-lichen planus overlap syndrome,lupus erythematosus panniculitis, tumid lupus erythematosus, verrucouslupus erythematosus cutaneous, systemic lupus erythematosus, subacutecutaneous lupus erythematosus, acute cutaneous lupus erythematosus,essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves'disease, lytic bone disease (e.g., multiple myeloma-induced lytice bonedisease), cystic fibrosis, age-related mascular degeneration (AMD; e.g.,wet AMD and dry AMD), liver fibrosis, pulmonary fibrosis,atherosclerosis, cardiac ischemia/reperfusion injury, heart failure,myocarditis, cardiac fibrosis, adverse myocardial remodeling,Guillain-Barre syndrome, Hashimoto's thyroiditis, psoriasis, psoriticarthritis, Crohn's Disease, ulcerative colitis, irritable bowel syndrome(IBS), inflammatory bowel disease (IBD), lupus nephritis, IgAnephropathy, diabetic kidney disease, minimal change disease (lipoidnephrosis), focal segmental glomerulosclerosis (FSGS), nephrogenicsystemic fibrosis (NSF), nephrogenic fibrosing dermopathy, fibrosingcholestatic hepatitis, eosinophilic fasciitis (Shulman's syndrome),scleromyxedema (popular mucinosis), scleroderma, lichensclerosusetatrophicus, POEMs syndrome (Crow-Fukase syndrome, Takatsukidisease or PEP syndrome), nephrotic syndrome, graft-versus-host-disease(GVHD), graft-versus-host-disease (GVHD) (from a transplant, such asblood, bone marrow, kidney, pancreas, liver, orthotopic liver, lung,heart, intestine, small intestine, large intestine, thymus, allogeneicstem cell, reduced-intensity allogeneic, bone, tendon, cornea, skin,heart valves, veins, arteries, blood vessels, stomach and testis). Thebispecific antibody, antibody or antigen-binding fragment made beadministered with another pharmaceutical agent, for example ananti-inflammatory agent, either in the same formulation or separately.

In another embodiment, the present invention provides a compositioncomprising an antibody, e.g., a bispecific antibody, as described hereinand a pharmaceutically acceptable carrier. A pharmaceutical compositioncomprising an antibody, e.g., a bispecific antibody, of the inventioncan be formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby the therapeutic proteins are combined in amixture with a pharmaceutically acceptable carrier. A composition issaid to be a “pharmaceutically acceptable carrier” if its administrationcan be tolerated by a recipient patient. Sterile phosphate-bufferedsaline is one example of a pharmaceutically acceptable carrier. Othersuitable carriers are well-known to those in the art. See, for example,Gennaro, ed., Remington's Pharmaceutical Sciences, 19th Edition, MackPublishing Company (1995).

For pharmaceutical use, an antibody, e.g., a bispecific antibody, of thepresent invention are formulated for parenteral, particularlyintravenous or subcutaneous, delivery according to conventional methods.Intravenous administration may be by bolus injection, controlledrelease, e.g., using mini-pumps or other appropriate technology, or byinfusion over a typical period of one to several hours. In general,pharmaceutical formulations will include an antibody, e.g., a bispecificantibody, of the invention in combination with a pharmaceuticallyacceptable carrier, such as saline, buffered saline, 5% dextrose inwater or the like. Formulations may further include one or moreexcipients, preservatives, solubilizers, buffering agents, albumin toprevent protein loss on vial surfaces, etc. When utilizing such acombination therapy, the antibodies, which include bispecificantibodies, may be combined in a single formulation or may beadministered in separate formulations. Methods of formulation are wellknown in the art and are disclosed, for example, in Gennaro, ed.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa.(1990), which is incorporated herein by reference. Therapeutic doseswill generally be in the range of 0.1 to 100 mg/kg of patient weight perday, preferably 0.5-20 mg/kg per day, with the exact dose determined bythe clinician according to accepted standards, taking into account thenature and severity of the condition to be treated, patient traits, etc.Determination of dose is within the level of ordinary skill in the art.More commonly, the antibodies will be administered over one week orless, often over a period of one to three days. Generally, the dosage ofadministered antibodies will vary depending upon such factors as thepatient's age, weight, height, sex, general medical condition andprevious medical history. Typically, it is desirable to provide therecipient with a dosage of antibodies which is in the range of fromabout 1 pg/kg to 10 mg/kg (amount of agent/body weight of patient),although a lower or higher dosage also may be administered ascircumstances dictate.

Administration of an antibody, e.g., bispecific antibody, of theinvention to a subject can be intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal,by perfusion through a regional catheter, or by direct intralesionalinjection. When administering therapeutic antibodies by injection, theadministration may be by continuous infusion or by single or multipleboluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase et al., “Oral Delivery of Microencapsulated Proteins”,in Sanders et al., eds., Protein Delivery: Physical Systems, pp.255-288, Plenum Press (1997)). The feasibility of an intranasal deliveryis exemplified by such a mode of insulin administration (see, forexample, Hinchcliffe et al., Adv. Drug Deliv. Rev., 35:199 (1999)). Dryor liquid particles comprising antibodies of the invention can beprepared and inhaled with the aid of dry-powder dispersers, liquidaerosol generators, or nebulizers (e.g., Pettit et al., TIBTECH, 16:343(1998); Patton et al., Adv. Drug Deliv. Rev., 35:235 (1999)). Thisapproach is illustrated by the AERX® diabetes management system, whichis a hand-held electronic inhaler that delivers aerosolized insulin intothe lungs. Studies have shown that proteins as large as 48,000 kDa havebeen delivered across skin at therapeutic concentrations with the aid oflow-frequency ultrasound, which illustrates the feasibility oftrascutaneous administration (Mitragotri et al., Science, 269:850(1995)). Transdermal delivery using electroporation provides anothermeans to administer a molecule having IL-17 and IL-23/p19 bindingactivity (Potts et al., Pharm. Biotechnol., 10:213 (1997)).

For purposes of therapy, compositions comprising an antibody, e.g., abispecific antibody, of the invention and a pharmaceutically acceptablecarrier are administered to a patient in a therapeutically effectiveamount. A combination of an antibody, e.g., a bispecific antibody, ofthe present invention and a pharmaceutically acceptable carrier is saidto be administered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient patient. For example, an agent used to treatinflammation is physiologically significant if its presence alleviatesthe inflammatory response. Effective treatment may be assessed in avariety of ways. In one embodiment, effective treatment is determined byreduced inflammation. In other embodiments, effective treatment ismarked by inhibition of inflammation. In still other embodiments,effective therapy is measured by increased well-being of the patientincluding such signs as weight gain, regained strength, decreased pain,thriving, and subjective indications from the patient of better health.

A pharmaceutical composition comprising an antibody, e.g., a bispecificantibody, of the invention can be furnished in liquid form, in anaerosol, or in solid form. Liquid forms, are illustrated by injectablesolutions and oral suspensions. Exemplary solid forms include capsules,tablets, and controlled-release forms. The latter form is illustrated byminiosmotic pumps and implants (Bremer et al., Pharm. Biotechnol.,10:239 (1997); Ranade, “Implants in Drug Delivery”, in Ranade et al.,eds., Drug Delivery Systems, pp. 95-123, CRC Press (1995); Bremer etal., “Protein Delivery with Infusion Pumps”, in Sanders et al., eds.,Protein Delivery: Physical Systems, pp. 239-254, Plenum Press (1997);Yewey et al., “Delivery of Proteins from a Controlled Release InjectableImplant”, in Sanders et al., eds., Protein Delivery: Physical Systems,pp. 93-117, Plenum Press (1997).

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis., 12(Suppl. 1):S61 (1993), Kim, Drugs, 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers”, inRanade et al., eds., Drug Delivery Systems, pp. 3-24, CRC Press (1995)).Liposomes are similar in composition to cellular membranes and as aresult, liposomes can be administered safely and are biodegradable.Depending on the method of preparation, liposomes may be unilamellar ormultilamellar, and liposomes can vary in size with diameters rangingfrom 0.02 μm to greater than 10 μm. A variety of agents can beencapsulated in liposomes: hydrophobic agents partition in the bilayersand hydrophilic agents partition within the inner aqueous space(s) (see,for example, Machy et al., Liposomes in Cell Biology and Pharmacology,John Libbey (1987), and Ostro et al., American J. Hosp. Pharm., 46:1576(1989)). Moreover, it is possible to control the therapeuticavailability of the encapsulated agent by varying liposome size, thenumber of bilayers, lipid composition, as well as the charge and surfacecharacteristics of the liposomes.

Liposomes can absorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci., 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta, 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta, 1068:133 (1991); Allen et al., Biochim. Biophys. Acta,1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent No. 04-244,018; Kato et al., Biol. Pharm. Bull.,16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull., 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato et al., Crit. Rev. Ther.Drug Carrier Syst., 14:287 (1997); Murahashi et al., Biol. Pharm. Bull.,20:259 (1997)). Similarly, Wu et al., Hepatology, 27:772 (1998), haveshown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull., 20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA, 94:11681 (1997)).Moreover, Geho et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev., 32:99(1998)). After plasma elimination of free antibody,streptavidin-conjugated liposomes are administered. In another approach,targeting antibodies are directly attached to liposomes (Harasym et al.,Adv. Drug Deliv. Rev., 32:99 (1998)).

Antibodies can be encapsulated within liposomes using standardtechniques of protein microencapsulation (see, for example, Anderson etal., Infect. Immun., 31:1099 (1981), Anderson et al., Cancer Res.,50:1853 (1990), and Cohen et al., Biochim. Biophys. Acta, 1063:95(1991), Alving et al. “Preparation and Use of Liposomes in ImmunologicalStudies”, in Gregoriadis, ed., Liposome Technology, 2nd Edition, Vol.III, p. 317, CRC Press (1993), Wassef et al., Meth. Enzymol., 149:124(1987)). As noted above, therapeutically useful liposomes may contain avariety of components. For example, liposomes may comprise lipidderivatives of poly(ethylene glycol) (Allen et al., Biochim. Biophys.Acta, 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz etal., Bioconjugate Chem., 6:332 (1995); Ranade, “Role of Polymers in DrugDelivery”, in Ranade et al., eds., Drug Delivery Systems, pp. 51-93, CRCPress (1995); Roskos et al., “Degradable Controlled Release SystemsUseful for Protein Delivery”, in Sanders et al., eds., Protein Delivery:Physical Systems, pp. 45-92, Plenum Press (1997); Bartus et al.,Science, 281:1161 (1998); Putney et al., Nature Biotechnology, 16:153(1998); Putney, Curr. Opin. Chem. Biol., 2:548 (1998)). Polyethyleneglycol (PEG)-coated nanospheres can also provide carriers forintravenous administration of therapeutic proteins (see, for example,Gref et al., Pharm. Biotechnol., 10:167 (1996).

The formulation can also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

In one embodiment, an antibody, e.g., a bispecific antibody, of theinvention is administered in combination therapy, i.e., combined withother agents, e.g., therapeutic agents, that are useful for treatingpathological conditions or disorders, such as autoimmune disorders andinflammatory diseases. The term “in combination” in this context meansthat the agents are given substantially contemporaneously, eithersimultaneously or sequentially. If given sequentially, at the onset ofadministration of the second compound, the first of the two compounds ispreferably still detectable at effective concentrations at the site oftreatment.

For example, the combination therapy can include one or more anantibodies, e.g., bispecific antibodies, of the invention coformulatedwith, and/or coadministered with, one or more additional therapeuticagents, e.g., one or more cytokine and growth factor inhibitors,immunosuppressants, anti-inflammatory agents, metabolic inhibitors,enzyme inhibitors, and/or cytotoxic or cytostatic agents, as describedin more detail below. Furthermore, one or more antibodies, e.g.,bispecific antibodies, described herein may be used in combination withtwo or more of the therapeutic agents described herein. Such combinationtherapies may advantageously utilize lower dosages of the administeredtherapeutic agents, thus avoiding possible toxicities or complicationsassociated with the various monotherapies.

Preferred therapeutic agents used in combination with an antibody, e.g.,bispecific antibody, of the invention are those agents that interfere atdifferent stages in an inflammatory response. In one embodiment, one ormore antibodies, e.g., bispecific antibodies, described herein may becoformulated with, and/or coadministered with, one or more additionalagents such as other cytokine or growth factor antagonists (e.g.,soluble receptors, peptide inhibitors, small molecules, ligand fusions);or antibodies or antigen binding fragments thereof that bind to othertargets (e.g., antibodies that bind to other cytokines or growthfactors, their receptors, or other cell surface molecules); andanti-inflammatory cytokines or agonists thereof. Nonlimiting examples ofthe agents that can be used in combination with the antibodies describedherein, include, but are not limited to, antagonists of one or moreinterleukins (ILs) or their receptors, e.g., antagonists of IL-1, IL-2,IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-18, IL-20, IL-21, IL-22and IL-31; antagonists of cytokines or growth factors or theirreceptors, such as tumor necrosis factor (TNF), LT, EMAP-II, GM-CSF, FGFand PDGF. Antibodies of the invention can also be combined withinhibitors of, e.g., antibodies to, cell surface molecules such as CD2,CD3, CD4, CD8, CD20 (e.g., the CD20 inhibitor rituximab (RITUXAN®)),CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, ortheir ligands, including CD154 (gp39 or CD40L), or LFA-1/ICAM-1 andVLA-4/VCAM-1 (Yusuf-Makagiansar et al., Med. Res. Rev., 22:146-167(2002)). Preferred antagonists that can be used in combination with oneor more antibodies, e.g., bispecific antibodies, described hereininclude antagonists of IL-1, IL-6, IL-12, TNF-alpha, IL-15, IL-18,IL-20, IL-22 and IL-31.

Examples of those agents include IL-12 antagonists, such as chimeric,humanized, human or in vitro-generated antibodies (or antigen bindingfragments thereof) that bind to IL-12 (preferably human IL-12), e.g.,the antibody disclosed in WO 00/56772; IL-12 receptor inhibitors, e.g.,antibodies to human IL-12 receptor; and soluble fragments of the IL-12receptor, e.g., human IL-12 receptor. Examples of IL-15 antagonistsinclude antibodies (or antigen binding fragments thereof) against IL-15or its receptor, e.g., chimeric, humanized, human or in vitro-generatedantibodies to human IL-15 or its receptor, soluble fragments of theIL-15 receptor, and IL-15-binding proteins. Examples of IL-18antagonists include antibodies, e.g., chimeric, humanized, human or invitro-generated antibodies (or antigen binding fragments thereof), tohuman IL-18, soluble fragments of the IL-18 receptor, and IL-18 bindingproteins (IL-18BP). Examples of IL-1 antagonists includeInterleukin-1-converting enzyme (ICE) inhibitors, such as Vx740, IL-1antagonists, e.g., IL-1 RA (anikinra, KINERET®, Amgen), sIL1RII(Immunex), and anti-IL-1 receptor antibodies (or antigen bindingfragments thereof).

Examples of TNF antagonists include chimeric, humanized, human or invitro-generated antibodies (or antigen binding fragments thereof) to TNF(e.g., human TNF-alpha), such as (HUMIRA®, D2E7, human TNF-alphaantibody), CDP-571/CDP-870/BAY-10-3356 (humanized anti-TNF-alphaantibody; Celltech/Pharmacia), cA2 (chimeric anti-TNF-alpha antibody;REMICADE®, Centocor); anti-TNF antibody fragments (e.g., CPD870);soluble fragments of the TNF receptors, e.g., p55 or p75 human TNFreceptors or derivatives thereof, e.g., 75 kdTNFR-IgG (75 kD TNFreceptor-IgG fusion protein, ENBREL®; Immunex), p55 kdTNFR-IgG (55 kDTNF receptor-IgG fusion protein (Lenercept)); enzyme antagonists, e.g.,TNF-alpha converting enzyme (TACE) inhibitors (e.g., an alpha-sulfonylhydroxamic acid derivative, and N-hydroxyformamide TACE inhibitor GW3333, -005, or -022); and TNF-bp/s-TNFR (soluble TNF binding protein).Preferred TNF antagonists are soluble fragments of the TNF receptors,e.g., p55 or p75 human TNF receptors or derivatives thereof, e.g., 75kdTNFR-IgG, and TNF-alpha converting enzyme (TACE) inhibitors.

In other embodiments, one or more antibodies, e.g., bispecificantibodies, described herein may be administered in combination with oneor more of the following: IL-13 antagonists, e.g., soluble IL-13receptors (sIL-13) and/or antibodies against IL-13; IL-2 antagonists,e.g., DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins, Seragen),and/or antibodies to IL-2R, e.g., anti-Tac (humanized anti-IL-2R,Protein Design Labs). Yet another combination includes one or moreantibodies, e.g., bispecific antibodies, of the invention, antagonisticsmall molecules, and/or inhibitory antibodies in combination withnondepleting anti-CD4 inhibitors (DEC-CE9.1/SB 210396; nondepletingprimatized anti-CD4 antibody; IDEC/SmithKline). Yet other preferredcombinations include antagonists of the costimulatory pathway CD80(B7.1) or CD86 (B7.2), including antibodies, soluble receptors orantagonistic ligands; as well as p-selectin glycoprotein ligand (PSGL),anti-inflammatory cytokines, e.g., IL-4 (DNAX/Schering); IL-10 (SCH52000; recombinant IL-10 DNAX/Schering); IL-13 and TGF-beta, andagonists thereof (e.g., agonist antibodies).

In other embodiments, one or more antibodies, e.g., bispecificantibodies, of the invention can be coformulated with, and/orcoadministered with, one or more anti-inflammatory drugs,immunosuppressants, or metabolic or enzymatic inhibitors. Nonlimitingexamples of the drugs or inhibitors that can be used in combination withthe antibodies described herein, include, but are not limited to, one ormore of: nonsteroidal anti-inflammatory drug(s) (NSAIDs), e.g.,ibuprofen, tenidap, naproxen, meloxicam, piroxicam, diclofenac, andindomethacin; sulfasalazine; corticosteroids such. as prednisolone;cytokine suppressive anti-inflammatory drug(s) (CSAIDs); inhibitors ofnucleotide biosynthesis, e.g., inhibitors of purine biosynthesis, folateantagonists (e.g., methotrexate(N-[4-[[(2,4-diamino-6-pteridinyl)methyl]methylamino]benzoyl]-L-glutamicacid); and inhibitors of pyrimidine biosynthesis, e.g., dihydroorotatedehydrogenase (DHODH) inhibitors. Preferred therapeutic agents for usein combination with one or more antibodies, e.g., bispecific antibodies,of the invention include NSAIDs, CSAIDs, (DHODH) inhibitors (e.g.,leflunomide), and folate antagonists (e.g., methotrexate).

Examples of additional inhibitors include one or more of:corticosteroids (oral, inhaled and local injection); immunosuppresants,e.g., cyclosporin, tacrolimus (FK-506); and mTOR inhibitors, e.g.,sirolimus (rapamycin—RAPAMUNE® or rapamycin derivatives, e.g., solublerapamycin derivatives (e.g., ester rapamycin derivatives, e.g.,CCI-779); agents which interfere with signaling by proinflammatorycytokines such as TNF-alpha or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAPkinase inhibitors); COX2 inhibitors, e.g., celecoxib, rofecoxib, andvariants thereof; phosphodiesterase inhibitors, e.g., R973401(phosphodiesterase Type IV inhibitor); phospholipase inhibitors, e.g.,inhibitors of cytosolic phospholipase 2 (cPLA2) (e.g., trifluoromethylketone analogs); inhibitors of vascular endothelial cell growth factoror growth factor receptor, e.g., VEGF inhibitor and/or VEGF-R inhibitor;and inhibitors of angiogenesis. Preferred therapeutic agents for use incombination with the antibodies of the invention are immunosuppresants,e.g., cyclosporin, tacrolimus (FK-506); mTOR inhibitors, e.g., sirolimus(rapamycin) or rapamycin derivatives, e.g., soluble rapamycinderivatives (e.g., ester rapamycin derivatives, e.g., CCI-779); COX2inhibitors, e.g., celecoxib and variants thereof; and phospholipaseinhibitors, e.g., inhibitors of cytosolic phospholipase 2 (cPLA2), e.g.,trifluoromethyl ketone analogs.

Additional examples of therapeutic agents that can be combined with anantibody, e.g., bispecific antibody, of the invention include one ormore of: 6-mercaptopurines (6-MP); azathioprine sulphasalazine;mesalazine; olsalazine; chloroquine/hydroxychloroquine (PLAQUENIL®);pencillamine; aurothiornalate (intramuscular and oral); azathioprine;coichicine; beta-2 adrenoreceptor agonists (salbutamol, terbutaline,salmeteral); xanthines (theophylline, aminophylline); cromoglycate;nedocromil; ketotifen; ipratropium and oxitropium; mycophenolatemofetil; adenosine agonists; antithrombotic agents; complementinhibitors; and adrenergic agents.

Nonlimiting examples of agents for treating or preventing arthriticdisorders (e.g., rheumatoid arthritis, inflammatory arthritis,rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis andpsoriatic arthritis), with which an antibody, e.g., bispecific antibody,of the invention may be combined include one or more of the following:IL-12 antagonists as described herein; NSAIDs; CSAIDs; TNFs, e.g.,TNF-alpha, antagonists as described herein; nondepleting anti-CD4antibodies as described herein; IL-2 antagonists as described herein;anti-inflammatory cytokines, e.g., IL-4, IL-10, IL-13 and TGF-alpha, oragonists thereof; IL-1 or IL-1 receptor antagonists as described herein;phosphodiesterase inhibitors as described herein; Cox-2 inhibitors asdescribed herein; iloprost: methotrexate; thalidomide andthalidomide-related drugs (e.g., Celgen); leflunomide; inhibitor ofplasminogen activation, e.g., tranexamic acid; cytokine inhibitor, e.g.,T-614; prostaglandin E1; azathioprine; an inhibitor of interleukin-1converting enzyme (ICE); zap-70 and/or 1 ck inhibitor (inhibitor of thetyrosine kinase zap-70 or 1 ck); an inhibitor of vascular endothelialcell growth factor or vascular endothelial cell growth factor receptoras described herein; an inhibitor of angiogenesis as described herein;corticosteroid anti-inflammatory drugs (e.g., SB203580); TNF-convertaseinhibitors; IL-11; IL-13; IL-17 inhibitors; gold; penicillamine;chloroquine; hydroxychloroquine; chlorambucil; cyclophosphamide;cyclosporine; total lymphoid irradiation; antithymocyte globulin;CD5-toxins; orally administered peptides and collagen; lobenzaritdisodium; cytokine regulating agents (CRAs) HP228 and HP466 (HoughtenPharmaceuticals, Inc.); ICAM-1 antisense phosphorothioateoligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.); solublecomplement receptor 1 (TP 10; T Cell Sciences, Inc.); prednisone;orgotein; glycosaminoglycan polysulphate; minocycline (MINOCIN®);anti-IL2R antibodies; marine and botanical lipids (fish and plant seedfatty acids); auranofin; phenylbutazone; meclofenamic acid; flufenamicacid; intravenous immune globulin; zileuton; mycophenolic acid(RS-61443); tacrolimus (FK-506); sirolimus (rapamycin); amiprilose(therafectin); cladribine (2-chlorodeoxyadenosine); and azaribine.Preferred combinations include one or more antibodies, e.g., bispecificantibodies, of the invention in combination with methotrexate orleflunomide, and in moderate or severe rheumatoid arthritis cases,cyclosporine.

Preferred examples of inhibitors to use in combination with one or moreantibodies, e.g., bispecific antibodies, of the invention to treatarthritic disorders include TNF antagonists (e.g., chimeric, humanized,human or in vitro-generated antibodies, or antigen binding fragmentsthereof, that bind to TNF; soluble fragments of a TNF receptor, e.g.,p55 or p75 human TNF receptor or derivatives thereof, e.g., 75kdTNFR-IgG (75 kD TNF receptor-IgG fusion protein, ENBREL®), p55 kD TNFreceptor-IgG fusion protein; TNF enzyme antagonists, e.g., TNF-alphaconverting enzyme (TACE) inhibitors); antagonists of IL-12, IL-15,IL-18, IL-22; T cell and B cell-depleting agents (e.g., anti-CD4 oranti-CD22 antibodies); small molecule inhibitors, e.g., methotrexate andleflunomide; sirolimus (rapamycin) and analogs thereof, e.g., CCI-779;cox-2 and cPLA2 inhibitors; NSAIDs; p38 inhibitors, TPL-2, Mk-2 and NFκBinhibitors; RAGE or soluble RAGE; P-selectin or PSGL-1 inhibitors (e.g.,small molecule inhibitors, antibodies thereto, e.g., antibodies toP-selectin); estrogen receptor beta (ERB) agonists or ERB—NFκBantagonists. Most preferred additional therapeutic agents that can becoadministered and/or coformulated with one or more antibodies, e.g.,bispecific antibodies, of the invention include one or more of: asoluble fragment of a TNF receptor, e.g., p55 or p75 human TNF receptoror derivatives thereof, e.g., 75 kdTNFR-IgG (75 kD TNF receptor-IgGfusion protein, ENBREL®); methotrexate, leflunomide, or a sirolimus(rapamycin) or an analog thereof, e.g., CCI-779.

Nonlimiting examples of agents for treating or preventing multiplesclerosis (e.g., relapsing-remitting multiple sclerosis,secondary-progressive multiple sclerosis, primary-progressive multiplesclerosis and progressive-relapsing multiple sclerosis) with one or moreantibodies, e.g., bispecific antibodies, of the invention can becombined include the following: interferons, e.g., interferon-alpha 1a(e.g., AVONEX®, Biogen) and interferon-1b (BETASERON®, Chiron/Berlex);Copolymer 1 (Cop-1; COPAXONE®, Teva Pharmaceutical Industries, Inc.);dimethyl fumarate (e.g., BG-12; Biogen); hyperbaric oxygen; intravenousimmunoglobulin; cladribine; TNF antagonists as described herein;corticosteroids; prednisolone; methylprednisolone; azathioprine;cyclophosphamide; cyclosporine; cyclosporine A, methotrexate;4-aminopyridine; and tizanidine. Additional antagonists that can be usedin combination with antibodies of the invention include antibodies to orantagonists of other human cytokines or growth factors, for example,TNF, LT, IL-1, IL-2, IL-6, EL-7, IL-8, IL-12 IL-15, IL-16, IL-18,EMAP-11, GM-CSF, FGF, and PDGF. Antibodies as described herein can becombined with antibodies to cell surface molecules such as CD2, CD3,CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or theirligands. One or more antibodies, e.g., bispecific antibodies, of theinvention may also be combined with agents, such as methotrexate,cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide,NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone,phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents,complement inhibitors, adrenergic agents, agents which interfere withsignaling by proinflammatory cytokines as described herein, IL-1bconverting enzyme inhibitors (e.g., Vx740), anti-P7s, PSGL, TACEinhibitors, T-cell signaling inhibitors such as kinase inhibitors,metalloproteinase inhibitors, sulfasalazine, azathloprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof, as described herein, andanti-inflammatory cytokines (e.g., IL-4, IL-10, IL-13 and TGF).

Preferred examples of therapeutic agents for multiple sclerosis (e.g.,relapsing-remitting multiple sclerosis, secondary-progressive multiplesclerosis, primary-progressive multiple sclerosis andprogressive-relapsing multiple sclerosis) with which the antibodies ofthe invention can be combined include dimethyl fumarate (e.g., BG-12;Biogen), interferon-beta, for example, IFN-beta-1a and IFN-beta-1b;COPAXONE®, corticosteroids, IL-1 inhibitors, TNF inhibitors, antibodiesto CD40 ligand and CD80, IL-12 antagonists.

Nonlimiting examples of agents for treating or preventing inflammatorybowel disease (e.g., Crohn's disease, ulcerative colitis) with which anantibody, e.g., bispecific antibody, of the invention can be combinedinclude the following: budenoside; epidermal growth factor;corticosteroids; cyclosporine; sulfasalazine; aminosalicylates;6-mercaptopurine; azathioprine; metronidazole; lipoxygenase inhibitors;mesalamine; olsalazine; balsalazide; antioxidants; thromboxaneinhibitors; IL-1 receptor antagonists; anti-IL-1 monoclonal antibodies;anti-IL-6 monoclonal antibodies (e.g., anti-IL-6 receptor antibodies andanti-IL-6 antibodies); growth factors; elastase inhibitors;pyridinyl-imidazole compounds; TNF antagonists as described herein;IL-4, IL-10, IL-13 and/or TGF.beta. cytokines or agonists thereof (e.g.,agonist antibodies); IL-11; glucuronide- or dextran-conjugated prodrugsof prednisolone, dexamethasone or budesonide; ICAM-1 antisensephosphorothioate oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals,Inc.); soluble complement receptor 1 (TP10; T Cell Sciences, Inc.);slow-release mesalazine; methotrexate; antagonists of plateletactivating factor (PAF); ciprofloxacin; and lignocaine.

Nonlimiting examples of agents for treating or preventing psoriasis withwhich an antibody, e.g., bispecific antibody, of the invention can becombined include the following: corticosteroids; vitamin D₃ and analogsthereof; retinoiods (e.g., soriatane); methotrexate; cyclosporine,6-thioguanine; Accutane; hydrea; hydroxyurea; sulfasalazine;mycophenolate mofetil; azathioprine; tacrolimus; fumaric acid esters;biologics such as AMEVIVE®, ENBREL®, HUMIRA®, Raptiva and REMICADE®,Ustekinmab, and XP-828L; phototherapy; and photochemotherapy (e.g.,psoralen and ultraviolet phototherapy combined).

Nonlimiting examples of agents for treating or preventing inflammatoryairway/respiratory disease (e.g., chronic obstructive pulmonarydisorder, asthma) with which an antibody, e.g., bispecific antibody, ofthe invention can be combined include the following: beta2-adrenoceptoragonists (e.g., salbutamol (albuterol USAN), levalbuterol, terbutaline,bitolterol); long-acting beta2-adrenoceptor agonists (e.g., salmeterol,formoterol, bambuterol); adrenergic agonists (e.g., inhaled epinephrineand ephedrine tablets); anticholinergic medications (e.g., ipratropiumbromide); combinations of inhaled steroids and long-actingbronchodilators (e.g., fluticasone/salmeterol (ADVAIR® in the UnitedStates, and Seretide in the United Kingdom)) or. budesonide/formoterol(SYMBICORT®)); inhaled glucocorticoids (e.g., ciclesonide,beclomethasone, budesonide, flunisolide, fluticasone, mometasone,triamcinolone); leukotriene modifiers (e.g., montelukast, zafirlukast,pranlukast, and zileuton); mast cell stabilizers (e.g., cromoglicate(cromolyn), and nedocromil); antimuscarinics/anticholinergics (e.g.,ipratropium, oxitropium, tiotropium); methylxanthines (e.g.,theophylline, aminophylline); antihistamines; IgE blockers (e.g.,Omalizumab); M.sub.3 muscarinic antagonists (anticholinergics) (e.g.,ipratropium, tiotropium); cromones (e.g., chromoglicate, nedocromil);zanthines (e.g., theophylline); and TNF antagonists (e.g., infliximab,adalimumab and etanercept).

In one embodiment, an antibody, e.g., bispecific antibody, of theinvention can be used in combination with one or more antibodiesdirected at other targets involved in regulating immune responses, e.g.,transplant rejection.

Nonlimiting examples of agents for treating or preventing immuneresponses with which an antibody, e.g., bispecific antibody, of theinvention can be combined include the following: antibodies againstother cell surface molecules, including but not limited to CD25(interleukin-2 receptor-a), CD11a (LFA-1), CD54 (ICAM-1), CD4, CD45,CD28/CTLA4 (CD80 (B7.1), e.g., CTLA4 Ig-abatacept (ORENCIA®)), ICOSL,ICOS and/or CD86 (B7.2). In yet another embodiment, an antibody of theinvention is used in combination with one or more generalimmunosuppressive agents, such as cyclosporin A or FK506.

In other embodiments, antibodies are used as vaccine adjuvants againstautoimmune disorders, inflammatory diseases, etc. The combination ofadjuvants for treatment of these types of disorders are suitable for usein combination with a wide variety of antigens from targetedself-antigens, i.e., autoantigens, involved in autoimmunity, e.g.,myelin basic protein; inflammatory self-antigens, e.g., amyloid peptideprotein, or transplant antigens, e.g., alloantigens. The antigen maycomprise peptides or polypeptides derived from proteins, as well asfragments of any of the following: saccharides, proteins,polynucleotides or oligonucleotides, autoantigens, amyloid peptideprotein, transplant antigens, allergens, or other macromolecularcomponents. In some instances, more than one antigen is included in theantigenic composition.

For example, desirable vaccines for moderating responses to allergens ina vertebrate host, which contain the adjuvant combinations of thisinvention, include those containing an allergen or fragment thereof.Examples of such allergens are described in U.S. Pat. No. 5,830,877 andPCT Publication No. WO 99/51259, which are hereby incorporated byreference in their entireties, and include pollen, insect venoms, animaldander, fungal spores and drugs (such as penicillin). The vaccinesinterfere with the production of IgE antibodies, a known cause ofallergic reactions. In another example, desirable vaccines forpreventing or treating disease characterized by amyloid deposition in avertebrate host, which contain the adjuvant combinations of thisinvention, include those containing portions of amyloid peptide protein(APP). This disease is referred to variously as Alzheimer's disease,amyloidosis or amyloidogenic disease. Thus, the vaccines of thisinvention include the adjuvant combinations of this invention plus Aβpeptide, as well as fragments of Aβ peptide and antibodies to Aβ peptideor fragments thereof.

In another embodiment, pharmaceutical compositions may be supplied as akit comprising a container that comprises an antibody, bispecificantibody or antigen-binding fragment of the invention. Antibodies, e.g.,bispecific antibodies, of the invention can be provided in the form ofan injectable solution for single or multiple doses, or as a sterilepowder that will be reconstituted before injection. Alternatively, sucha kit can include a dry-powder disperser, liquid aerosol generator, ornebulizer for administration of the antibody, e.g., bispecific antibody.Such a kit may further comprise written information on indications andusage of the pharmaceutical composition. Moreover, such information mayinclude a statement that the antibody composition is contraindicated inpatients with known hypersensitivity to IL-17 and IL-23.

In a further embodiment, the invention provides an article ofmanufacture, comprising: (a) a composition of matter comprising anantibody, bispecific antibody or antigen-binding fragment as describedherein; (b) a container containing said composition; and (c) a labelaffixed to said container, or a package insert included in saidcontainer referring to the use of said antibody in the treatment of animmune related disease.

In another aspect, the composition comprises a further activeingredient, which may, for example, be a further antibody or ananti-inflammatory, cytotoxic or chemotherapeutic agent. Preferably, thecomposition is sterile.

The antibodies, bispecific antibodies and antigen-binding fragments asdescribed herein are also useful to prepare medicines and medicamentsfor the treatment of immune-related and inflammatory diseases, includingfor example, multiple sclerosis (MS) (e.g., relapsing-remitting multiplesclerosis, secondary-progressive multiple sclerosis, primary-progressivemultiple sclerosis and progressive-relapsing multiple sclerosis),irritable bowel syndrome (IBS), inflammatory bowel disease (IBD) such asulcerative colitis and Crohn's disease, atopic dermatitis, contactdermatitis, systemic sclerosis, systemic lupus erythematosus (SLE),antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis(AAV), giant cell arteritis, multiple sclerosis (MS), colitis,endotoxemia, arthritis, rheumatoid arthritis (RA), osteoarthritis,Sjögren's syndrome, psoriasis, psoriatic arthritis, adult respiratorydisease (ARD), septic shock, multiple organ failure, inflammatory lunginjury such as idiopathic pulmonary fibrosis, asthma, chronicobstructive pulmonary disease (COPD), airway hyper-responsiveness,chronic bronchitis, allergic asthma, eczema, Helicobacter pyloriinfection, intraabdominal adhesions and/or abscesses as results ofperitoneal inflammation (e.g., from infection, injury, etc.), nephroticsyndrome, idiopathic demyelinating polyneuropathy, Guillain-Barresyndrome, organ allograft rejection, graft vs. host disease (GVHD),lupus nephritis, IgA nephropathy, diabetic kidney disease, minimalchange disease (lipoid nephrosis), focal segmental glomerulosclerosis(FSGS), nephrogenic systemic fibrosis (NSF), nephrogenic fibrosingdermopathy, fibrosing cholestatic hepatitis, eosinophilic fasciitis(Shulman's syndrome), scleromyxedema (popular mucinosis), scleroderma,lichen sclerosusetatrophicus, POEMs syndrome (Crow-Fukase syndrome,Takatsuki disease or PEP syndrome), nephrotic syndrome,graft-versus-host-disease (GVHD), graft-versus-host-disease (GVHD) (froma transplant, such as blood, bone marrow, kidney, pancreas, liver,orthotopic liver, lung, heart, intestine, small intestine, largeintestine, thymus, allogeneic stem cell, reduced-intensity allogeneic,bone, tendon, cornea, skin, heart valves, veins, arteries, bloodvessels, stomach and testis), lytic bone disease (e.g., multiplemyeloma-induced lytice bone disease), cystic fibrosis, age-relatedmascular degeneration (AMD; e.g., wet AMD and dry AMD), liver fibrosis,pulmonary fibrosis, atherosclerosis, cardiac ischemia/reperfusioninjury, heart failure, myocarditis, cardiac fibrosis, adverse myocardialremodeling, transplant rejection, streptococcal cell wall (SCW)-inducedarthritis, gingivitis/periodontitis, herpetic stromal keratitis,gluten-sensitive enteropathy restenosis, Kawasaki disease, and immunemediated renal diseases. In a specific aspect, such medicines andmedicaments comprise a therapeutically effective amount of a bispecificantibody, antibody or antigen-binding fragment of the invention with apharmaceutically acceptable carrier. In an embodiment, the admixture issterile.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (e.g., GENBANK®amino acid and nucleotide sequence submissions) cited herein areincorporated by reference. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. The invention isnot limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

The invention is further illustrated by the following non-limitingexamples.

Example 1 Humanization of a Murine Anti-Human IL-17A/F Dual SpecificAntibody

Selection of Hybridoma Clones and Variable Region Identification

Recombinant human proteins IL-17A, IL-17A/F, and IL-17F were producedusing an HEK293 transient expression system at ZymoGenetics Inc., aBristol-Myers Squibb Company (Seattle, Wash., USA). BALB/c mice (CharlesRiver Laboratories, Wilmington, Mass.) were immunized and boosted withrecombinant human IL-17F conjugated to BSA followed by immunizationswith recombinant human IL-17A conjugated to BSA. The mice with seracontaining the highest anti-IL-17F and anti-IL-17A antibody bindingactivity were given a final pre-fusion boost of IL-17F. Four days later,the splenocytes and lymph node cells were fused with Ag8.653 myelomacells to generate antibody producing hybridomas. Hybridoma culturesupernatants were screened for IL-17F and IL-17A binding by plate basedELISA and IL-17F and IL-17A neutralization in the IL-17A/F cell-basedassay. Hybridoma cells corresponding to the supernatant sample thatbound and neutralized both IL-17F and IL-17A were cloned in order toisolate a monoclonal hybridoma, 339.15.3.5 (designated as 339.15)producing the neutralizing monoclonal antibody of interest. Hybridoma339.15 was isotyped using the ISOSTRIP® Mouse Monoclonal AntibodyIsotyping Kit (Roche, Indianapolis, Ind., USA) and RNA was isolatedusing the QIAGEN® RNeasy kit (Qiagen, Valencia, Calif., USA). Variableregions were cloned using the SMART RACE cDNA Amplification Kit(Clontech, Mountain View, Calif., USA), utilizing 5′ RACE technology andgene specific 3′ primers designed to mouse constant region sequences.Heavy and light variable region sequences were cloned using the TOPO® TACloning Kit for Sequencing (Invitrogen, Carlsbad, Calif., USA). Genesequences were verified by comparing the sequence to the N-terminalamino acid sequencing performed on antibody purified from hybridoma339.15.

Variable region sequences were cloned from 339.15.3.5, 339.15.5.3 and339.15.3.6 and were shown to contain the exact same variable regionsequences. The sequence from 339.15.3.5 was used for subsequenthumanization, and the 339.15.3.6 hybridoma clone was deposited on Nov.7, 2006, with the American Type Tissue Culture Collection (ATCC, 10801University Blvd, Manassas, Va. 20110-2209) patent depository as originaldeposits under the Budapest Treaty and was given ATCC® Patent DepositDesignation PTA-7988. Hybridoma clone 339.15.3.6 (ATCC® Patent DepositDesignation PTA-7988) and 339.15.5.3 (ATCC Patent Deposit DesignationPTA-7987) are also disclosed, for example, in U.S. Pat. Nos. 7,790,163,7,910,703 and 8,333,968.

Molecular Modeling of Chimeric and Humanized Anti-Human IL-17A/FVariable Region Sequences

All variable region models were constructed and viewed using the MOESoftware Suite, Version 2008 (Chemical Computing Group, Montreal,Canada).

Anti-Human IL-17A/F Humanized Antibody Design

Murine complementarity determining regions (CDR) were grafted onto humangermline framework sequences. The sequences were compared to germlineamino acid sequences in V-Base (MRC, Center for Protein Engineering,UK). One germline gene was chosen for the variable heavy region, VH1-03.Several germline genes were chosen for the variable light region; VKVIA26, VKI A20, VKVI A14, VKIII L6, and VKI L14. The VKVI germline genefamily showed the highest homology to the murine sequence, however,being an under represented germline family in the human antibodyrepertoire, other germline families with high homology were alsoconsidered. Murine Kabat defined CDR regions were grafted on human Kabatdefined framework regions for both the heavy and light chains.

Construction, Expression, and Purification of Humanized Anti-IL-17A/FAntibodies

Humanized variable region sequences were ordered from GeneART, Inc.(GeneART, Inc. Burlingame, Calif., USA). Humanized and murine variableregion sequences were fused to human kappa constant region (SEQ IDNO:10) or IgG1.1 (SEQ ID NO:11), an effector minus variant of wild-typeIgG1 that has mutations resulting in the reduction of Fc γ receptor Ibinding and ability to fix complement (Gross et al., Immunity,15:289-302 (2001)), utilizing overlap PCR (Horton et al., Gene, 77:61-68(1989)) and/or restriction enzyme cloning into pTT5, an HEK293-6Etransient expression vector (NCR Biotechnology Research Institute,Ottawa, ON, CAN). All constructs were expressed using the mod 2610(ATGCGGCGGAGAGGCTGGTCCTGGATCTTCCTGTTTCTGCTGAGCGGAACAG CCGGCGTGCTGAGC,SEQ ID NO:30) signal sequence, although any nucleic acid sequence thatencodes the amino acid sequence MRRRGWSWIFLFLLSGTAGVLS (SEQ ID NO:31)may be used. The HEK293-6E suspension cells were transfected withexpression constructs using polyethylenimine reagent and cultivated inF17 medium (Invitrogen, Grand Island, N.Y., USA) with the addition of 5mM L-glutamine and 25 μg/mL G418. After 24 hours, 1/40th volume of 20%Tryptone NI (Organotechnie SAS, La Courneuve, F R) was added. Atapproximately 120 hours post transfection, conditioned media washarvested and passed through a 0.2 μm filter. Protein was purified fromthe filtered conditioned media using a combination of Mab Select SuReAffinity Chromatography (GE Healthcare, Piscataway, N.J., USA) andSUPERDEX® 200 Size Exclusion Chromatography (GE Healthcare, Piscataway,N.J., USA). Content was estimated by absorbance at UV-A280 nm andquality evaluated by analytical size exclusion high performance liquidchromatography, SDS PAGE, and western blot.

Anti-Human IL-17A/F Humanization Panel Bioassay Activity; NIH/3T3/KZ170NE-κB Luciferase Reporter Assay to Measure Human IL-17A, IL-17A/F, andIL-17F Activity by NE-κB Induction

A murine fibroblast cell line (NIH/3T3, ATCC® #CRL-1658) was stablytransfected with an NF-κB luciferase reporter designated KZ170 andcloned out. NIH/3T3/KZ170 clone 1 cells were seeded at 10,000 cells/wellin plating media (DMEM plus 3% FBS, 1 mM sodium pyruvate, 2 mML-glutamine (HyClone Laboratories, South Logan, Utah)) in 96-well, whiteopaque, solid bottom luciferase plates (Corning Incorporated, Corning,N.Y.) and incubated overnight at 37° C., 5% CO₂. The following dayserial dilutions of recombinant human IL-17A, IL-17A/F, or IL-17F(ZymoGenetics, A Bristol-Myers Squibb Company, Seattle, Wash., USA) weremade up in assay media (DMEM plus 0.5% BSA, 1 mM sodium pyruvate, 2 mML-glutamine, 10 mM HEPES (HyClone Laboratories, South Logan, Utah)) andadded to the plates containing the cells and incubated together at 37°C., 5% CO₂ for 4 hours. Additionally the assay was used to measureneutralization of human IL-17A, IL-17A/F and IL-17F activity. A halfmaximal concentration (EC₅₀, effective concentration at 50 percent) ofhuman IL-17A, IL-17A/F or IL-17F was combined with serial dilutions ofanti-human IL-17A/F antibodies described herein in assay media and addedto the plates containing the cells and incubated together at 37° C., 5%CO₂ for 4 hours. Following incubation the media was removed and cellslysed before being read on the Berthold Centro XS³ Luminometer (BertholdTechnologies, Wildbad, Germany) using flash substrate (PromegaCorporation, Madison, Wis.) according to manufacturer's instructions.Increases in mean fluorescence intensity (via activation of the NF-κBluciferase reporter) were indicative of a human IL-17A, IL-17A/F, IL-17Freceptor-ligand interaction. Decreases in mean fluorescence intensitywere indicative of neutralization of the human IL-17A, IL-17A/F, IL-17Freceptor-ligand interaction. IC₅₀ (inhibitory concentration at 50percent) values were calculated using GraphPad Prism 4 software(GraphPad Software, Inc., San Diego Calif.) for each anti-human IL-17A/Fantibody.

Anti-Human IL-17A/F Humanization CDR Grafted and Chimeric Panel BioassayActivity; NIH/3T3/KZ170 NE-κB Luciferase Reporter Assay Results

IL-17A, IL-17A/F and IL-17F induce activation of the NF-κB luciferasereporter in a dose dependent manner with an EC₅₀ concentrationdetermined to be 0.15 nM for IL-17A, 0.50 nM for IL-17A/F and 0.50 nMfor IL-17F. Tables 1 and 2 present example IC₅₀ data for theanti-IL-17A/F antibodies described herein.

TABLE 1 IL- 17A/ IL- VH VL F 17F MVC# MVC# IL-17A IC₅₀ IC₅₀ Name SEQ IDNO: SEQ ID NO: IC₅₀ nM nM nM Chimeric Ms VH VR370 Ms VL VR371 11 0.300.26 339.15 MVC823 MVC824 SEQ ID NO: 32 SEQ ID NO: 34 339-07 VR370e3VH1- Ms VL >600 31 5.5 03 MVC824 MVC840 SEQ ID NO: 34 SEQ ID NO: 36339-08 Ms VH VR370 VR371e3 VKVI 1.5 0.96 0.81 MVC823 A26 SEQ ID NO: 32MVC841 SEQ ID NO: 38 339-02 Ms VH VR370 VR371e2 VKI 1.1 0.80 0.79 MVC823A20 SEQ ID NO: 32 MVC717 SEQ ID NO: 40 339-01 Ms VH VR370 VR371e1 VKVI9.6 0.26 0.20 MVC823 A14 SEQ ID NO: 32 MVC716 SEQ ID NO: 42 339-09 Ms VHVR370 VR371e4 VKIII 7.2 0.20 0.21 MVC823 L6 SEQ ID NO: 32 MVC842 SEQ IDNO: 44 339-32 Ms VH VR370 VR371e10 VKI 7.0 1.5 0.35 MVC823 L14 SEQ IDNO: 32 MVC856 SEQ ID NO: 46 339-33 VR370e3 VH1- VR371e3 VKVI >600 9.71.7 03 A26 MVC840 MVC841 SEQ ID NO: 36 SEQ ID NO: 38 339-126 VR370e3VH1- VR371e2 VKI >600 24 1.6 03 A20 MVC840 MVC717 SEQ ID NO: 36 SEQ IDNO: 40Anti-Human IL-17A/F Humanization CDR Grafted with Framework BackMutation Panel Bioassay Activity Table: NIH/3T3/KZ170 NF-κB LuciferaseReporter Assay Results

TABLE 2 IL- 17A/ IL- VH VL F 17F MVC# MVC# IL-17A IC₅₀ IC₅₀ Name SEQ IDNO: SEQ ID NO: IC₅₀ nM nM nM 339-35 VR370e4 NKSH VR371e3 VKVI >600 2.40.64 MVC850 A26 SEQ ID NO: 48 MVC841 SEQ ID NO: 38 339-71 VR370e41 KALVVR371e3 VKVI 16 0.37 0.20 MVC869 A26 SEQ ID NO: 50 MVC841 SEQ ID NO: 38339-37 VR370e6 SF VR371e3 VKVI >600 20 3.5 MVC852 A26 SEQ ID NO: 52MVC841 SEQ ID NO: 38 339-38 VR370e7 VR371e3 VKVI 8.2 0.27 0.27 NKSH KALVA26 MVC853 MVC841 SEQ ID NO: 54 SEQ ID NO: 38 339-39 VR370e8 VR371e3VKVI 7.6 0.23 0.25 NKSH KALV SF A26 MVC854 MVC841 SEQ ID NO: 56 SEQ IDNO: 38 339-127 VR370e4 NKSH VR371e2 VKI A20 190 3.4 0.50 MVC850 MVC717SEQ ID NO: 48 SEQ ID NO: 40 339-128 VR370e41 KALV VR371e2 VKI A20 5.10.41 0.25 MVC869 MVC717 SEQ ID NO: 50 SEQ ID NO: 40 339-105 VR370e6 SFVR371e2 VKI A20 >600 23 2.6 MVC852 MVC717 SEQ ID NO: 52 SEQ ID NO: 40339-125 VR370e7 VR371e2 VKI A20 1.5 0.81 0.83 NKSH KALV MVC717 MVC853SEQ ID NO: 40 SEQ ID NO: 54 339-104 VR370e8 VR371e2 VKI A20 1.5 0.830.83 NKSH KALV SF MVC717 MVC854 SEQ ID NO: 40 SEQ ID NO: 56 339-134VR370e96 VR371e2 VKI A20 1.4 0.26 0.24 NK KALV MVC717 MVC978 SEQ ID NO:40 SEQ ID NO: 58Anti-Human IL-17A/F Humanization Panel Biacore Activity; Measurement ofBinding Affinities to Human IL-17A, IL-17A/F, and IL-17F Via SurfacePlasmon Resonance (Biacore)

Humanized anti-human IL-17A/F monoclonal antibodies were evaluated fortheir binding affinity to human IL-17A, human IL-17A/F, and human IL-17Fusing surface plasmon resonance.

Kinetic rate constants and equilibrium dissociation constants weremeasured for the interaction of the humanized anti-human IL-17A/Fantibodies with human IL-17A, IL-17A/F, and IL-17F via surface plasmonresonance. The association rate constant (k_(a) (M⁻¹s⁻¹)) is a valuethat reflects the rate of the antigen-antibody complex formation. Thedissociation rate constant (k_(d) (s⁻¹)) is a value that reflects thestability of this complex. By dividing the dissociation rate constant bythe association rate constant (k_(d)/k_(a)) the equilibrium dissociationconstant (K_(D) (M)) is obtained. This value describes the bindingaffinity of the interaction. Antibodies with similar K_(D) can havewidely variable association and dissociation rate constants.Consequently, measuring both the k_(a) and k_(d) of antibodies helps tomore uniquely describe the affinity of the antibody-antigen interaction.

Binding kinetics and affinity studies were performed on a BIACORE® T100system (GE Healthcare, Piscataway, N.J.). Methods for the BIACORE® T100were programmed using BIACORE® T100 Control Software, v2.0. For theseexperiments, the humanized anti-human IL-17A/F antibodies were capturedonto a CM4 sensor chip via either goat anti-human IgG Fc-gamma antibody(Jackson ImmunoResearch, West Grove, Pa.) or goat anti-mouse IgGFc-gamma antibody (Jackson ImmunoResearch). Binding experiments with theIL-17 molecules were performed at 25° C. in a buffer of 10 mM HEPES, 150mM NaCl, 3 mM EDTA, 0.05% Surfactant P20 (GE Healthcare), 1 mg/mL bovineserum albumin, pH 7.4.

The capture antibody, goat anti-human IgG Fc-gamma, was diluted toconcentration of 20 μg/mL in 10 mM sodium acetate pH 5.0, and thencovalently immobilized to all four flow cells of a CM4 sensor chip usingamine coupling chemistry (EDC:NHS). After immobilization of theantibody, the remaining active sites on the flow cell were blocked with1 M ethanolamine. A capture antibody density of approximately 5000 RUwas obtained. The humanized anti-human IL-17A/F antibodies were capturedonto flow cell 2, 3, or 4 of the CM4 chip at a density ranging from60-150 RU. Capture of the test antibodies to the immobilized surface wasperformed at a flow rate of 10 μL/min. The BIACORE® instrument measuresthe mass of protein bound to the sensor chip surface, and thus, captureof the test antibody was verified for each cycle. Serial dilutions ofhuman IL-17A, IL-17A/F, or IL-17F (ZymoGenetics, A Bristol-Myers, SquibbCompany, Seattle, Wash., USA) were prepared from 100 nM-0.032 nM (1:5serial dilutions). The serial dilutions were injected over the surfaceand allowed to specifically bind to the test antibody captured on thesensor chip. Duplicate injections of each antigen concentration wereperformed with an association time of 7 minutes and dissociation time of15 minutes. Kinetic binding studies were performed with a flow rate of50 μL/min. In between cycles, the flow cell was washed with 20 mMhydrochloric acid to regenerate the surface. This wash step removed boththe captured test antibody and any bound antigen from the immobilizedantibody surface. The test antibody was subsequently captured again inthe next cycle.

Data was compiled using the BIACORE® T100 Evaluation software (version2.0). Data was processed by subtracting reference flow cell and blankinjections. Baseline stability was assessed to ensure that theregeneration step provided a consistent binding surface throughout thesequence of injections. Duplicate injection curves were checked forreproducibility. Based on the binding of the bivalent IL-17 molecules toa bivalent antibody, the bivalent analyte binding interaction model wasdetermined to be appropriate for interactions with the IL-17 molecules.The bivalent analyte model is previously described in West, A. P. etal., Biochemistry, 39:9698-9708 (2000); and West, A. P. et al., J. Mol.Biol., 313:385-397 (2001). An affinity constant (k_(D1)) under thebivalent analyte model may be calculated from the ratio of rateconstants (k_(d1)/k_(a1)) as determined by surface plasmon resonance.The reference subtracted binding curves were globally fit to theappropriate binding model with a multiple Rmax and with the RI set tozero. The data fit well to the binding models with good agreementbetween the experimental and theoretical binding curves. The chi² andstandard errors associated the fits were low. There was no trending inthe residuals.

Anti-Human IL-17A/F Humanization Panel Biacore Activity

The results of the binding experiments with human IL-17A, IL-17A/F, andIL-17F are in Tables 3, 4, and 5 respectively.

Anti-Human IL-17A/F Humanized Antibodies Binding Affinity for IL-17A

TABLE 3 k_(a1) k_(d1) Name (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M) Mouse 4.E+06 7.E−032.E−09 339.15 339-02 2.E+06 6.E−03 3.E−09 SEQ ID NO: 32 SEQ ID NO: 40Chimeric 3.E+06 1.E−02 4.E−09 339.15 SEQ ID NO: 32 SEQ ID NO: 34 339-384.E+06 5.E−03 1.E−9 SEQ ID NO: 54 SEQ ID NO: 38 339-125 2.E+06 3.E−031.E−9 SEQ ID NO: 54 SEQ ID NO: 40 339-134 2.E+06 3.E−03 1.E−9 SEQ ID NO:58 SEQ ID NO: 40Anti-IL-17A/F Humanized Antibodies Binding Affinity for IL-17A/F

TABLE 4 k_(a1) k_(d1) Name (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M) Mouse 1.E+06 2.E−042.E−10 339.15 339-02 Not Determined SEQ ID NO: 32 SEQ ID NO: 40 ChimericNot Determined 339.15 SEQ ID NO: 32 SEQ ID NO: 33 339-38 1.E+06 4.E−044.E−10 SEQ ID NO: 54 SEQ ID NO: 40 339-125 2.E+06 6.E−04 3.E−10 SEQ IDNO: 54 SEQ ID NO: 40 339-134 2.E+06 5.E−04 2.E−10 SEQ ID NO: 58 SEQ IDNO: 40Anti-IL-17A/F Humanized Antibodies Binding Affinity for IL-17F

TABLE 5 k_(a1) k_(d1) Name (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M) Mouse 2.E+06 1.E−045.E−11 339.15 339-02 1.E+06 5.E−04  4E−10 SEQ ID NO: 32 SEQ ID NO: 40Chimeric 1.E+06 5.E−04  4E−10 339.15 SEQ ID NO: 32 SEQ ID NO: 34 339-382.E+06 6.E−04 3.E−10 SEQ ID NO: 54 SEQ ID NO: 40 339-125 2.E+06 2.E−041.E−10 SEQ ID NO: 54 SEQ ID NO: 40 339-134 2.E+06 2.E−04 1.E−10 SEQ IDNO: 58 SEQ ID NO: 40

Example 2 7B7 Antibody Selection and Hybridoma Generation

Epitope Binning Approach by Surface Plasmon Resonance Technology (UsingBiacore) to Group Antibodies Based on their Binding and BlockingProperties as Shown in FIG. 11.

Antibodies were grouped and selected based on their ability to:

1. Specifically bind p19 subdomain only of IL-23;

2. Specifically block only IL-23 receptor (IL-23R) and not block IL-12receptor (IL-12R); and

3. Not compete with any antibody that could bind specifically to p40subdomain of IL-23.

Materials such as antibodies with previously known selectivity for p19or p40 subdomains of IL-23 and IL-23R or IL-12R were all chosen to becoated on a BIACORE® CM5 chip. The coating density varied between 500 to8000 Resonance Units (RUs). Antibodies that were to be binned weretitrated serially (1:2 or 1:3) to 8 concentrations, from startingconcentrations that ranged from 10 to 100 μg/mL in a 96-well ELISAplate. To each of the well, 10 nM of IL-23 antigen was added. Theantibodies on plate were allowed to form a complex with antigen andreach equilibrium overnight at 4° C. The complexes were injected overthe CM5 chip at a flow rate of 20 μL/min for two minutes. The signal, asbinding resonance units (RUs) at end of two minutes was noted. Theantibody-antigen complex was able complete or not compete with thematerial that was coated on the chip. If the antibody in complex withantigen was able to compete with the material on chip, with increasingconcentration of the antibody, the binding RU decreased and if it didnot compete, the binding RU increased. Based on this observation, allanti-IL23 antibodies were binned according to their bindingselectivities and competing abilities.

Transgenic HCo12 J/K HUMAB® Mice from the Medarex Colonies in Milpitas,Calif. were Immunized with Recombinant Human IL-23-his in RIBI Adjuvant.

Sera from immunized mice were tested for expression of IL-23 specificantibodies by a modified indirect dual ELISA. Briefly, microtiter plates(COSTAR®, 96-well flat bottom, #9018) were coated with mouse anti-hisprotein at 2.5 μg/ml in PBS, 50 μl/well, incubated at 4° C. overnight,and then blocked with 1% BSA in PBS. HuIL-23 at 2.5 μg/ml or HuIL-12 wasadded to plates for capture at 50 μl/well and incubated at roomtemperature for one hour. Plates were washed with PBS Tween, anddilutions of sera were added and incubated for 1 hour. The plates werewashed with PBS-Tween and incubated with goat-anti-human gamma heavychain conjugated with HRP (Jackson ImmunoResearch Cat. 109-036-098) for1 hour. After 3× washing, the plates were developed with ABTS (Moss, CAT#ABTS-1000) substrate and OD's analyzed at 415 nm. Data were analyzedand expressed as serum titer which is defined as the highest dilution ofserum which results in an antigen positive signal of at least twicebackground. Mouse 215094 was selected for hybridoma generation basedupon relatively high titers on IL-23 with lower cross reactivity toIL-12 when compared to other mice in the cohort (see Table 6).

TABLE 6 Serum Titers Mouse ID Genotype Hu IL23-his Hu IL12-his 215088HCo12:01[J/K] >109, 350 >109, 350 215090 HCo12:01[J/K] >109, 350 >109,350 215092 HCo12:01[J/K] >109, 350 >109, 350 215094 HCo12:01[J/K] >109,350    12, 150 215096 HCo12:01[J/K] >109, 350    36, 450 215098HCo12:01[J/K]    36, 450    1, 350 215089 HCo12:01[J/K] >109, 350    1,350 215091 HCo12:01[J/K] >109, 350 >109, 350 215093 HCo12:01[J/K] >109,350 4,050 215095 HCo12:01[J/K] >109, 350 >109, 350 215097HCo12:01[J/K] >109, 350 >109, 350 215099 HCo12:01[J/K] 12150    12, 150

The genotype of Mouse 215094 is provided below in Table 7.

TABLE 7 Mouse 215094 Genotype Mouse ID Sex Date of birth Genotype 215094M Oct. 11, 2009 HCo12(15087)+{circumflex over ( )}; JHD++; JKD++;KCo5(9272)+{circumflex over ( )};

The spleen from mouse 215094 was used to generate hybridomas with mousemyeloma cells (ATCC CRL-1580) by electric field based electrofusionusing a CytoPulse large chamber cell fusion electroporation device in aprocedure designated fusion 2378.

Conditioned media from the resulting hybridomas were initially screenedfor expression of human IgG γ/κ in a standard automated assay followedby ELISA for IL-23 binding with a counter screen ELISA on IL-12 toidentify specific clones as previously described. Hybridoma selectioncriteria for testing were samples with OD's greater than 1.5 on huIL23plates and less than 0.15 on huIL12.

Fusion 2378 generated total of 827 human IgG positive hybridomas ofwhich, 128 were IL-23 specific. Hybridoma 7B7 was selected for furthertesting based on its strong binding to IL-23 and lack of crossreactivity to IL-12, when compared to anti-p19 and anti-p40 positivecontrol antibodies; an example of hybridomas, including 7B7 selected byELISA is given in FIG. 7. The isotype of subclone 7B7.D4 was confirmedas human IgG1, kappa by ELISA.

Hybridoma conditioned medium from all IL-23p-19 specific MAbs werescreened for IL-23 neutralizing activity in a cell-based assay. Kit225,a human T-cell line established from a patient with T-cell chroniclymphocytic leukemia, have been shown to respond to IL-23 with dosedependant STAT3 phosphorylation (pSTAT3). Human IL-23 at EC₅₀ with andwithout the addition of hybridoma conditioned medium or a controlneutralizing anti-p19 antibody was used to stimulate cells for 15minutes. Cells were lysed and inhibition of IL-23 dependant STAT3phosphorylation was assessed by ELISA (Cell Signaling Technology,PATHSCAN® Cat #7300) where reduced O.D. indicates reduced levels ofpSTAT3. Hybridoma 7B7 was selected for sub-cloning and furthercharacterization based upon the potent neutralization of IL-23 signalingobserved in the Kit225 assay and as shown in FIG. 8.

Using assays similar to those described above, selective binding ofIL-23 and neutralization of IL-23 signaling was demonstrated for the 7B7subclone 1413.2378.7B7.D4.H2 which was subsequently submitted forsequencing (IL-23p19 7B7 heavy chain variable domain is shown in SEQ IDNO:7, and the light chain variable domain is shown in SEQ ID NO:9).

Example 3 Generation of Anti-Human IL-23/IL-17A/F Bispecific Antibodies

Construction and Expression of Mammalian Anti-Human IL-23/IL-17A/FBispecific Molecules

Partial or whole genes were synthesized at GeneART, Inc. (GeneART, Inc.Burlingame, Calif., USA) or GenScript (GenScript, Piscataway, N.J., USA)and inserted into pTT5, an HEK293-6E transient expression vector (NCRBiotechnology Research Institute, Ottawa, ON, Canada) via restrictionenzyme cloning. MVC1059 (SEQ ID NO:62), and MVC1061 (SEQ ID NO:60) wereordered as complete constructs from GenScript (GenScript, Piscataway,N.J., USA). All constructs were expressed using the mod 2610 (SEQ IDNO:30) signal sequence. The biAbFabL is a bispecific antibody whichcontains a whole antibody with a C-terminal Fab unit of the second armof the bispecific attached via a linker (e.g., 10 mer G₄S) and utilizesa common light chain (see FIG. 2). The taFab is a bispecific antibodywhich contains a whole antibody with an N-terminal Fab unit of thesecond arm of the bispecific attached via a linker, such as(Gly₄Ser₁)_(x), wherein x is 1, 2 or 3, and the linker of SEQ ID NO:12.As with the heavy chain portion, there are two light chains for each armof the bispecific attached via a linker, such as (Gly₄Ser₁)_(x), whereinx is 1, 2 or 3, and the linker of SEQ ID NO:12 (see FIG. 3). TheHeterodimeric Fc is a bispecific antibody that resembles a traditionalantibody, however, contains two different heavy chains which associatethrough an electrostatic complementarity association in the C_(H3)region. The Heterodimeric Fc utilizes a common light chain (see FIG. 4).Heavy chain and light chain constant regions include, IgG1.1 (SEQ IDNO:11, which may be encoded by SEQ ID NO:82), IgG1.1f without aC-terminal Lysine (SEQ ID NO:127), IgG1.1f with a C-terminal Lysine (SEQID NO:128), human kappa constant region (SEQ ID NO:10, which may beencoded by SEQ ID NO:83), or IgG4.1 (SEQ ID NO:8). The IgG4 heavy chainconstant domain may be a variant of wild-type IgG4 that has a mutationin the hinge region, S228P (EU index numbering system) or S241P (Kabatnumbering system). Changing the serine at 241 (Kabat) to proline (foundat that position in IgG1 and IgG2) in a mouse/human chimeric heavy chainleads to the production of a homogeneous antibody and abolishes theheterogeneity. Further, the variant IgG4 has significantly extendedserum half-life and shows an improved tissue distribution compared tothe original chimeric IgG4. Angal et al., Molecular Immunology,30(1):105-108 (1993); Schuurman et al., Molecular Immunology, 38:1-8(2001); Lewis et al., Molecular Immunology, 46:3488-3494 (2009).

Transformation of electrocompetent E. coli host cells (DH10B) wasperformed using 1 μl of the yeast DNA preparation and 20 μl of E. colicells. The cells were electropulsed at 2.0 kV, 25 μF, and 400 ohms.Following electroporation, 600 μl SOC (2% BACTO® Tryptone (Difco,Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10mM MgCl₂, 10 mM MgSO₄, 20 mM glucose) was added and the cells wereplated in 50 μl and 550 μl aliquots on two LB AMP plates (LB broth(Lennox), 1.8% BACTO® Agar (Difco), 100 mg/L Ampicillin).

Five colonies from each construct were subjected to sequence analysis.One clone containing the correct sequence was selected. DNA sequencingwas performed using ABI PRISM® BigDye Terminator v3.1 Cycle SequencingKit (Applied Biosystems, Foster City, Calif.). Sequencing reactions werepurified using Edge BioSystems Preforma Centriflex Gel FiltrationCartridges (Gaithersburg, Md.) and run on an Applied Biosystems 3730 DNAAnalyzer (Applied Biosystems, Foster City, Calif.). Resultant sequencedata was assembled and edited using SEQUENCHER® v4.6 software (GeneCodesCorporation, Ann Arbor, Mich.). One clone containing the correctsequence was selected and large-scale plasmid DNA was isolated using acommercially available kit (QIAGEN® Plasmid Mega Kit, Qiagen, Valencia,Calif.) according to manufacturer's instructions.

The HEK293-6E suspension cells were transfected with expressionconstructs using polyethylenimine reagent and cultivated in F17 medium(Invitrogen, Grand Island, N.Y., USA) with the addition of 5 mML-glutamine and 25 μg/mL G418. After 24 hours, 1/40th volume of 20%Tryptone NI (Organotechnie SAS, La Courneuve, F R) was added. Atapproximately 120 hours post transfection, conditioned media washarvested and passed through a 0.2 μm filter. Protein was purified fromthe filtered conditioned media using a combination of Mab Select SuReAffinity Chromatography (GE Healthcare, Piscataway, N.J., USA) andSUPERDEX® 200 Size Exclusion Chromatography (GE Healthcare, Piscataway,N.J., USA). Content was estimated by absorbance at UV-A280 nm andquality evaluated by analytical size exclusion high performance liquidchromatography, SDS PAGE, and western blot.

Anti-Human IL-23/IL-17A/F Bispecific Antibody Composition

A whole antibody and its modular components is depicted in FIG. 1. ThebiAbFabL format is depicted in FIG. 2. The taFab format is depicted inFIG. 3. The heterodimeric Fc format is depicted in FIG. 4. The VCVFcformat is depicted in FIG. 5. The VCDFc format is depicted in FIG. 6.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;NIH/3T3/KZ170 NE-κB Luciferase Reporter Assay to Measure Human IL-17A,IL-17A/F, and IL-17F Activity by NE-κB Induction

The material and methods for this assay are described in Example 1hereinabove.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Baf3/huIL-23Rα/huIL-12R/β1 Transfectants Phospho-STAT3 Assay to MeasureHuman IL-23 Activity by Phospho-STAT3 Induction

A murine bone marrow derived cell line (Baf3) was stably transfectedwith human IL-23Rα and human IL-12Rβ1 and cloned.Baf3/huIL-23Rα/huIL-12Rβ1 clone 6 cells were washed three times withassay media (RPMI 1640 plus 10% fetal bovine serum, 2 mM L-Glutamine, 1mM Sodium Pyruvate (HyClone Laboratories, South Logan, Utah), and 2 μMβ-Mercaptoethanol (Sigma-Aldrich, St. Louis, Mo.)) before being platedout at 50,000 cells/well in 96-well, round-bottom tissue culture plates.Serial dilutions of recombinant human IL-23 (ZymoGenetics, ABristol-Myers Squibb Company, Seattle Wash., USA) were made up in assaymedia and added to the plates containing the cells and incubatedtogether at 37° C., 5% CO₂ for 15 minutes. Additionally the assay wasalso used to measure neutralization of IL-23 activity. A half maximalconcentration (EC₅₀, effective concentration at 50 percent) of IL-23 wascombined with serial dilutions of anti-human IL-23/IL-17A/F antibodiesdescribed herein and incubated together at 37° C., 5% CO₂ for 15 minutesin assay media prior to addition to cells. Following pre-incubation,treatments were added to the plates containing the cells and incubatedtogether at 37° C., 5% CO₂ for 15 minutes. Following incubation, cellswere washed with ice-cold wash buffer and put on ice to stop thereaction according to manufacturer's instructions (BIO-PLEX® Cell LysisKit, Bio-Rad Laboratories, Hercules, Calif.). Cells were then spun downat 2000 rpm at 4° C. for 5 minutes prior to dumping the media. FiftyμL/well lysis buffer was added to each well; lysates were pipetted upand down five times while on ice, then agitated on a plate shaker for 20minutes at 300 rpm and 4° C. Plates were centrifuged at 3200 rpm at 4°C. for 20 minutes. Supernatants were collected and transferred to a newmicro titer plate for storage at −80° C.

Capture beads (BIO-PLEX® Phospho-STAT3 Assay, Bio-Rad Laboratories) werecombined with 50 μL of 1:1 diluted lysates and added to a 96-well filterplate according to manufacturer's instructions (BIO-PLEX® PhosphoproteinDetection Kit, Bio-Rad Laboratories). The aluminum foil-covered platewas incubated overnight at room temperature, with shaking at 300 rpm.The plate was transferred to a microtiter vacuum apparatus and washedthree times with wash buffer. After addition of 25 μL/well detectionantibody, the foil-covered plate was incubated at room temperature for30 minutes with shaking at 300 rpm. The plate was filtered and washedthree times with wash buffer. Streptavidin-PE (50 μL/well) was added,and the foil-covered plate was incubated at room temperature for 15minutes with shaking at 300 rpm. The plate was filtered and washed threetimes with bead resuspension buffer. After the final wash, beads wereresuspended in 125 μL/well of bead suspension buffer, shaken for 30seconds, and read on an array reader (BIO-PLEX® 100, Bio-RadLaboratories) according to the manufacturer's instructions. Data wasanalyzed using analytical software (BIO-PLEX® Manager 4.1, Bio-RadLaboratories). Increases in the level of the phosphorylated STAT3transcription factor present in the lysates were indicative of an IL-23receptor-ligand interaction. Decreases in the level of thephosphorylated STAT3 transcription factor present in the lysates wereindicative of neutralization of the IL-23 receptor-ligand interaction.IC₅₀ (inhibitory concentration at 50 percent) values were calculatedusing GraphPad Prism 4 software (GraphPad Software, Inc., San DiegoCalif.) for each anti-human IL-23/IL-17A/F antibody.

Anti-Human IL-23/IL-17A/F Bispecific Antibody Bioassay Activity;NIH/3T3/KZ170 NF-κB Luciferase Reporter Assay andBaf3/huIL-23Rα/huIL-12Rβ1 Transfectants Phospho-STAT3 Assay Results

Human IL-17A, IL-17A/F and IL-17F induce activation of the NF-κBluciferase reporter in a dose dependent manner with an EC₅₀concentration determined to be 0.33 nM for IL-17A, 1 nM for IL-17A/F and1 nM for IL-17F and IL-23 induces STAT3 phosphorylation in a dosedependent manner with an EC₅₀ concentration determined to be 0.02 nM.The IC₅₀ data for the anti-human IL-23/IL-17A/F bispecific antibodies isshown below in Table 8.

Anti-Human IL-23/17A/F Bispecific Antibody Table

TABLE 8 Heavy Chain IL- MVC# Light Chain IL- 17F IL-23 SEQ ID MVC#IL-17A 17A/F IC₅₀ IC₅₀ Name NO: SEQ ID NO: IC₅₀ nM IC₅₀ nM nM nM 339-134MVC978 MVC717 1.3 0.27 0.24 Not mAb IgG1.1 SEQ ID SEQ ID Done NO: 64 NO:66 IL23.6 (7B7) MVC1003 MVC1002 Not Not Not 0.014 mAb IgG1.1 SEQ ID SEQID Done Done Done NO: 68 NO: 17* 23/17bAb1 MVC1006 MVC1002 0.064 0.760.96 0.015 IgG1.1 SEQ ID SEQ ID NO: 28* NO: 17* 23/17bAb2 MVC1007MVC1002 0.052 0.43 0.44 0.041 IgG1.1 SEQ ID SEQ ID NO: 18* NO: 17*23/17bAb3 MVC1036 MVC1002 0.022 0.20 0.23 0.012 IgG4.1 SEQ ID SEQ ID NO:74 NO: 17* 23/17bAb4 MVC1037 MVC1002 0.035 0.18 0.87 0.048 IgG4.1 SEQ IDSEQ ID NO: 29* NO: 17* 23/17taFab1 MVC1008 MVC 1009 1.5 3.9 2.3 0.018IgG1.1 SEQ ID SEQ ID NO: 76 NO: 78 23/17hetero1 MVC1059 MVC1002 0.340.78 0.33 0.060 IgG1.1 SEQ ID SEQ ID NO: 62 NO: 17* MVC1060 SEQ ID NO:64 23/17hetero2 MVC1061 MVC1002 0.71 2.33 0.96 0.055 IgG1.1 SEQ ID SEQID NO: 60 NO: 17* MVC1062 SEQ ID NO: 80 *The amino acid sequence of SEQID NO: 17 may be encoded by the sequence of SEQ ID NO: 70; the aminoacid sequence of SEQ ID NO: 28 may be encoded by the sequence of SEQ IDNO: 71; the amino acid sequence of SEQ ID NO: 18 may be encoded by thesequence of SEQ ID NO: 72; the amino acid sequence of SEQ ID NO: 29 maybe encoded by the sequence of SEQ ID NO: 75.Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Primary Human SAEC Assay to Measure Human IL-17A, IL-17AF, and IL-17FActivity by G-CSF Induction

Primary human small airway epithelial cells (SAEC) were seeded at 8,000cells/well in Small Airway Epithelial Growth Medium (SAGM) (cells andmedia: Lonza, Walkersville, Md.) in 96-well flat bottom tissue cultureplates (Corning Incorporated, Corning, N.Y.) and incubated overnight at37° C., 5% CO₂. The following day serial dilutions of human IL-17A,IL-17A/F, or IL-17F (ZymoGenetics, A Bristol-Myers Squibb Company,Seattle, Wash., USA) were made up in SAGM media and added to the platescontaining the cells and incubated together at 37° C., 5% CO₂ for 24hours. Additionally the assay was used to measure neutralization ofIL-17A, IL-17A/F and IL-17F activity. A half maximal concentration(EC₅₀, effective concentration at 50 percent) of IL-17A, IL-17A/F orIL-17F was combined with serial dilutions of anti-human IL-23/IL-17A/Fbispecific antibodies described herein in SAGM media and added to theplates containing the cells and incubated together at 37° C., 5% CO₂ for24 hours. After incubation the supernatants were spun down, collectedand frozen at −80° C. until ready to process. Human G-CSF protein levelsin the supernatants were measured using a commercial bead based humanG-CSF cytokine ELISA according to manufactures instructions(Procarta/Affymetrix, Santa Clara, Calif.). Increases in human G-CSFlevels in the supernatant were indicative of a human IL-17A, IL-17A/F,IL-17F receptor-ligand interaction. Decreases in human G-CSF levels inthe supernatant were indicative of neutralization of the human IL-17A,IL-17A/F, IL-17F receptor-ligand interaction. IC₅₀ (inhibitoryconcentration at 50 percent) values were calculated using GraphPad Prism4 software (GraphPad Software, Inc., San Diego Calif.) for eachanti-human IL-23/IL-17A/F bispecific antibody.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Primary Human SAEC Assay Results

Human IL-17A, IL-17A/F and IL-17F induce human G-CSF production in adose dependent manner with an EC₅₀ concentration determined to be 0.03nM for IL-17A, 3 nM for IL-17A/F and 3 nM for IL-17F. Bispecificantibodies tested include 23/17bAb1 (SEQ ID NO:28 and SEQ ID NO:17),23/17bAb2 (SEQ ID NO:18 and SEQ ID NO:17), 23/17bAb3 (SEQ ID NO:74 andSEQ ID NO:17), 23/17bAb4 (SEQ ID NO:29 and SEQ ID NO:17). The humanizedanti-human IL-17A/F antibody 339-134 mAb (SEQ ID NO:65 and SEQ ID NO:67)was also tested. The IC₅₀ data for the anti-human IL-23/IL-17A/Fbispecific antibodies is shown below in Table 9. This data indicatesthat the anti-IL-23/IL-17A/F bispecific antibodies inhibit human IL-17A,IL-17A/F, IL-17F mediated IL-6 production were equally potent.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Primary Human Fibroblast Assay to Measure Human IL-17A, IL-17A/F, andIL-17F Activity by IL-6 Induction

A primary human fibroblast cell line (HFFF2, Cat #86031405, HealthProtection Agency Culture Collections, Porton Down Salisbury, UK) wasseeded at 5,000 cells/well in assay media (DMEM plus 10% FBS and 2 mML-glutamine (HyClone Laboratories, South Logan, Utah)) in 96-well flatbottom plates (Corning Incorporated, Corning, N.Y.) and incubatedovernight at 37° C., 5% CO₂. The following day serial dilutions ofrecombinant human IL-17A, IL-17AF, or IL-17F (ZymoGenetics, ABristol-Myers Squibb Company, Seattle, Wash. 98117) were made up inassay media and added to the plates containing the cells and incubatedtogether at 37° C., 5% CO₂ for 24 hours. Additionally the assay was usedto measure neutralization of human IL-17A, IL-17A/F and IL-17F activity.A half maximal concentration (EC₅₀, effective concentration at 50percent) of human IL-17A, IL-17A/F or IL-17F was combined with serialdilutions of anti-human IL-23/IL-17A/F antibodies described herein inassay media and added to the plates containing the cells and incubatedtogether at 37° C., 5% CO₂ for 24 hours. After incubation thesupernatants were spun down, collected and frozen at −80° C. until readyto process. Human IL-6 protein levels in the supernatants were measuredusing a commercial bead based human IL-6 cytokine ELISA according tomanufactures instructions (Bio-Rad Laboratories, Hercules, Calif.).Increases in human IL-6 levels in the supernatant were indicative of ahuman IL-17A, IL-17A/F, IL-17F receptor-ligand interaction. Decreases inhuman IL-6 levels in the supernatant were indicative of neutralizationof the human IL-17A, IL-17A/F, IL-17F receptor-ligand interaction. IC₅₀(inhibitory concentration at 50 percent) values were calculated usingGraphPad Prism 4 software (GraphPad Software, Inc., San Diego Calif.)for each anti-human IL-23/IL-17A/F bispecific antibody.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Primary Human Fibroblast Assay Results

Human IL-17A, IL-17A/F and IL-17F induce human IL-6 production in a dosedependent manner with an EC₅₀ concentration determined to be 0.08 nM forIL-17A, 25 nM for IL-17AF and 25 nM for IL-17F. Bispecific antibodiestested include 23/17bAb1 (SEQ ID NO:28 and SEQ ID NO:17), 23/17bAb2 (SEQID NO:18 and SEQ ID NO:17), 23/17bAb3 (SEQ ID NO:74 and SEQ ID NO:17),23/17bAb4 (SEQ ID NO:29 and SEQ ID NO:17). Humanized anti-human IL-17A/Fantibody 339-134 mAb (SEQ ID NO:64 and SEQ ID NO:66) was also tested.The IC₅₀ data for the anti-human IL-23/IL-17A/F bispecific antibodies isshown below in Table 9. These data indicate that the anti-humanIL-23/IL-17A/F bispecific antibodies inhibit human IL-17A, IL-17A/F,IL-17F mediated IL-6 production were equally potent.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Murine Splenocyte Assay to Measure Human IL-23 Activity by Murine IL-17Aand IL-17F Induction

A single cell suspension of murine splenocytes was prepared from wholespleens harvested from BALB/c mice. After red blood cell lysis with ACKbuffer (0.010 M KHCO₃, 0.0001 M EDTA, 0.150 M NH₄Cl, pH 7.2) splenocyteswere washed and resuspended in assay media (RPMI 1640 plus 10% FBS,non-essential amino acids, 1 mM Sodium Pyruvate, 2 mM L-glutamine, 10 mMHEPES, 100 units/mL Pen/Strep (HyClone Laboratories, South Logan, Utah),50 μM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, Mo.), and 50 ng/mlhuman IL-2 (R&D Systems, Minneapolis, Minn.)). Splenocytes were seededat 500,000 cells per well in 96-well round bottom plates. Serialdilutions of recombinant human IL-23 (BDC 50220AN087 heterodimermaterial) were made up in assay media and added to the plates containingthe cells and incubated together at 37° C., 5% CO₂ for 24 hours.Additionally the assay was also used to measure neutralization of humanIL-23 activity. A half maximal concentration (EC₅₀, effectiveconcentration at 50 percent) of human IL-23 was combined with serialdilutions of anti-human IL-23/IL-17A/F bispecific antibodies describedherein and incubated together at 37° C., 5% CO₂ for 15 minutes in assaymedia prior to addition to cells. Following pre-incubation, treatmentswere added to the plates containing the cells and incubated together at37° C., 5% CO₂ for 24 hours. After incubation the supernatants were spundown, collected and frozen at −80° C. until ready to process. Theprotein levels of murine IL-17A and IL-17F in the supernatants weremeasured using commercial plate based murine IL-17A and IL-17F ELISA'saccording to manufacturer's instructions (eBiosciences, San Diego,Calif.). Increases in murine IL-17A and IL-17F levels in the supernatantwere indicative of an IL-23 receptor-ligand interaction. Decreases inmurine IL-17A and IL-17F levels in the supernatant were indicative ofneutralization of the IL-23 receptor-ligand interaction. IC₅₀(inhibitory concentration at 50 percent) values were calculated usingGraphPad Prism 4 software (GraphPad Software, Inc., San Diego Calif.)for each anti-human IL-23/IL-17A/F bispecific antibody.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Murine Splenocyte Assay Results

Human IL-23 induced murine IL-17A and IL-17F in a dose dependent mannerwith an EC₅₀ concentration determined to be 0.01 nM. Bispecificantibodies tested include 23/17bAb1 (SEQ ID NO:28 and SEQ ID NO:17),23/17bAb2 (SEQ ID NO:18 and SEQ ID NO:17), 23/17bAb3 (SEQ ID NO:74 andSEQ ID NO:17), 23/17bAb4 (SEQ ID NO:29 and SEQ ID NO:17). The anti-humanIL-23.6 (7B7) mAb (SEQ ID NO:68 and SEQ ID NO:17) was also tested. TheIC₅₀ data for the anti-human IL-23/IL-17A/F bispecific antibodies isshown below in Table 9. This data indicates that the anti-humanIL-23/IL-17A/F bispecific antibodies inhibit human IL-23 induced murineIL-17A and IL-17F production.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Primary Human T Cell Phospho-STAT3 Assay to Measure Human IL-23 Activityby Phospho-STAT3 Induction

Leukopheresis PBMC: Normal human donors (ZymoGenetics' normal donorpool) were selected at random and were voluntarily apheresed at theFHCRC (Seattle, Wash.). The leukopheresis PBMC were delivered toZymoGenetics in a sterile blood-collection bag. The cells were pouredinto a sterile 500 mL plastic bottle, diluted to 400 ml, with roomtemperature PBS plus 1 mM EDTA (HyClone Laboratories, South Logan, Utah)and transferred to 250 mL conical tubes. The 250 mL tubes werecentrifuged at 1500 rpm for 10 minutes to pellet the cells. The cellsupernatant was then removed and discarded. The cell pellets were thencombined and suspended in 400 mL PBS plus 1 mM EDTA. The cell suspension(25 mL/tube) was overlaid onto FICOLL® (20 mL/tube) in 50 mL conicaltubes (total of 16 tubes). The tubes were centrifuged at 2000 rpm for 20minutes at room temperature. The interface layer (“buffy coat”)containing the white blood cells and residual platelets was collected,pooled and washed repeatedly with PBS plus 1 mM EDTA until the majorityof the platelets had been removed. The white blood cells were thensuspended in 100 mL of ice-cold Cryopreservation medium (70% RPMI 1640,20% FCS, 10% DMSO (HyClone Laboratories)) and distributed into sterilecryovials (1 mL cells/vial). The cryovials placed in a −80° C. freezerfor 24 hours before transfer to a liquid-nitrogen freezer. The whiteblood-cell yield from a typical apheresis is 0.5-1.0×10¹⁰ cells.Apheresis cells processed in this manner contain T cells, B cells, NKcells, monocytes and dendritic cells.

Preparation of Activated T Cells:

T cells must be activated in order to express the IL-12 receptor and beable to respond to IL-12 and IL-23. Cryopreserved leukopheresis PBMCwere thawed, transferred to a sterile 50 mL conical tube, and washedwith 50 mL of warm assay media (RPMI 1640 plus 10% FBS (HyCloneLaboratories)) and incubated in a 37° C. water bath for 1 hour to allowthe cells to recover. The cells were then centrifuged and thecell-supernatant discarded. The cell pellet was resuspended in assaymedia and distributed into sterile 162 cm² tissue culture flasks at2×10⁷ cells per flask in 90 mL assay media containing 5 μg/mL PHA-M(Roche, Basel, Switzerland). The cells were then cultured at 37° C. in ahumidified incubator for a total of 5 days. The cells were “rested” byharvesting on the afternoon of day 4, replacing the culture medium withfresh assay media without PHA and returning to the incubator for theremainder of the 5 day culture period.

Phospho-STAT3 Assay:

Activated human T cells were harvested on day 5 of culture andresuspended in fresh assay media and were plated out at 2×10⁵ cells/wellin U-bottom 96-well plates. Serial dilutions of recombinant human IL-23(BDC 50220AN087 heterodimer material) were made up in assay media andadded to the plates containing the cells and incubated together at 37°C., 5% CO₂ for 15 minutes. Additionally the assay was also used tomeasure neutralization of IL-23 activity. A half maximal concentration(EC₅₀, effective concentration at 50 percent) of IL-23 was combined withserial dilutions of anti-human IL-23/IL-17AF antibodies described hereinand incubated together at 37° C., 5% CO₂ for 15 minutes in assay mediaprior to addition to cells. Following pre-incubation, treatments wereadded to the plates containing the cells and incubated together at 37°C., 5% CO₂ for 15 minutes. Following incubation, cells were washed withice-cold wash buffer and put on ice to stop the reaction according tomanufacturer's instructions (BIO-PLEX® Cell Lysis Kit, Bio-RadLaboratories, Hercules, Calif.). Cells were then spun down at 2000 rpmat 4° C. for 5 minutes prior to dumping the media. Fifty μL/well lysisbuffer was added to each well; lysates were pipetted up and down fivetimes while on ice, then agitated on a plate shaker for 20 minutes at300 rpm and 4° C. Plates were centrifuged at 3200 rpm at 4° C. for 20minutes. Supernatants were collected and transferred to a new microtiter plate for storage at −80° C.

Capture beads (BIO-PLEX® Phospho-STAT3 Assay, Bio-Rad Laboratories) werecombined with 50 μL of 1:1 diluted lysates and added to a 96-well filterplate according to manufacturer's instructions (BIO-PLEX® PhosphoproteinDetection Kit, Bio-Rad Laboratories). The aluminum foil-covered platewas incubated overnight at room temperature, with shaking at 300 rpm.The plate was transferred to a microtiter vacuum apparatus and washedthree times with wash buffer. After addition of 25 μL/well detectionantibody, the foil-covered plate was incubated at room temperature for30 minutes with shaking at 300 rpm. The plate was filtered and washedthree times with wash buffer. Streptavidin-PE (50 μL/well) was added,and the foil-covered plate was incubated at room temperature for 15minutes with shaking at 300 rpm. The plate was filtered and washed threetimes with bead resuspension buffer. After the final wash, beads wereresuspended in 125 μL/well of bead suspension buffer, shaken for 30seconds, and read on an array reader (BIO-PLEX® 100, Bio-RadLaboratories) according to the manufacturer's instructions. Data wasanalyzed using analytical software (BIO-PLEX® Manager 4.1, Bio-RadLaboratories). Increases in the level of the phosphorylated STAT3transcription factor present in the lysates were indicative of an IL-23receptor-ligand interaction. Decreases in the level of thephosphorylated STAT3 transcription factor present in the lysates wereindicative of neutralization of the IL-23 receptor-ligand interaction.IC₅₀ (inhibitory concentration at 50 percent) values were calculatedusing GraphPad Prism 4 software (GraphPad Software, Inc., San DiegoCalif.) for each anti-human IL-23/IL-17A/F bispecific antibody.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Activity;Primary Human T Cell Phospho-STAT3 Assay Results

Human IL-23 induces STAT3 phosphorylation in a dose dependent mannerwith an EC₅₀ concentration determined to be 0.02 nM. Bispecificantibodies tested include 23/17bAb1 (SEQ ID NO:28 and SEQ ID NO:17),23/17bAb2 (SEQ ID NO:18 and SEQ ID NO:17), 23/17bAb3 (SEQ ID NO:74 andSEQ ID NO:17), 23/17bAb4 (SEQ ID NO:29 and SEQ ID NO:17). The anti-humanIL-23.6 (7B7) mAb (SEQ ID NO:68 and SEQ ID NO:17) was also tested. TheIC₅₀ data for the anti-human IL-23/IL-17A/F antibodies is shown below inTable 9.

Anti-Human IL-23/IL-17A/F Bispecific Antibody Bioassay Activity; PrimaryHuman SAEC Assay, Primary Human Fibroblast Assay, Murine SplenocyteAssay and Primary Human T Cell Phospho-STAT3 Assay Results

TABLE 9 23/17bAb1 23/17bAb2 23/17bAb3 23/17bAb4 339-134 IL23.6(7B7)IgG1.1 IgG1.1 IgG4.1 IgG4.1 mAbIgG1.1 mAbIgG1.1 SEQ ID NO: 28 SEQ ID NO:18 SEQ ID NO: 74 SEQ ID NO: 29 SEQ ID NO: 64 SEQ ID NO: 68 Profile SEQID NO: 17 SEQ ID NO: 17 SEQ ID NO: 17 SEQ ID NO: 17 SEQ ID NO: 66 SEQ IDNO: 17 Cellular Potency IL-17A <0.5 pM <0.5 pM <0.5 pM ≦0.5 pM 0.5 nMNot Done Hu. primary EC₅₀ = epithelial cells 0.03 nM (SAEC) IL-17AF 1.4nM 1.3 nM 0.5 nM 1.4 nM 1.3 nM Not Done IC₅₀ EC₅₀ = 3 nM IL-17F 0.8 nM1.6 nM 1.0 nM 1.3 nM 1.1 nM Not Done EC₅₀ = 3 nM Cellular Potency IL-17A0.07 nM 0.07 nM 0.03 nM 0.1 nM 0.9 nM Not Done Hu. primary EC₅₀ =fibroblast cells 0.08 nM (HFFF) IL-17AF 17 nM 12 nM 9.4 nM 9.1 nM  13 nMNot Done IC₅₀ EC₅₀ = 25 nM IL-17F 19 nM 15 nM 10 nM 12 nM  15 nM NotDone EC₅₀ = 25 nM Cellular potency IL-23 0.1 nM 0.06 nM 0.1 nM 0.08 nMNot Done 0.09 nM Murine EC₅₀ = splenocyte assay 0.01 nM IC₅₀ Cellularpotency IL-23 0.04 nM 0.05 nM 0.04 nM 0.1 nM Not Done 0.04 nM Primary Tcell EC₅₀ = assay IC₅₀ 0.02 nM

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Co-Binding Activity;Primary Human Fibroblast Assay to measure the inhibition of humanIL-17A, IL-7A/F, or IL-F while simultaneously bound to human IL-23. ThePrimary Human T Cell Phospho-STAT3 Assay to measure the inhibition ofhuman IL-23 while simultaneously bound to human IL-17A, IL-7A/F, orIL-17F.

The primary human fibroblast assay was run in the presence of excessamounts of IL-23 at 30 nM. The primary human T cell phospho-STAT3 assaywas run in the presence of excess amounts of IL-17A, IL-17A/F, IL-17F at30 nM.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Bioassay Co-BindingResults

Bispecific antibodies tested include 23/17bAb1 (SEQ ID NO:28 and SEQ IDNO:17), 23/17bAb2 (SEQ ID NO:18 and SEQ ID NO:17), 23/17bAb3 (SEQ IDNO:74 and SEQ ID NO:17), 23/17bAb4 (SEQ ID NO:29 and SEQ ID NO:17), and23/17taFab1 (SEQ ID NO:76 and SEQ ID NO:78). The anti-humanIL-23/IL-17A/F bispecific antibodies when examined in the presence ofhuman IL-23 did not interfere with human IL-17A, IL-17A/F, IL-17Finhibition. The anti-human IL-23/IL-17A/F bispecific antibodies whenexamined in the presence of human IL-17A, IL-17A/F, IL-17F did notinterfere with human IL-23 inhibition.

Measurement of Binding Affinities of Anti-Human IL-23/IL-17A/FBispecific Antibodies to Human IL-17A, IL-17A/F, IL-17F, and Human IL-23Via Surface Plasmon Resonance (Biacore)

Anti-human IL-23/IL-17A/F bispecific antibodies were evaluated for theirbinding affinity to human IL-17A, human IL-17A/F, human IL-17F, andhuman IL-23 using surface plasmon resonance.

Kinetic rate constants and equilibrium dissociation constants weremeasured for the interaction of the anti-human IL-23/IL-17A/F bispecificantibodies with human IL-17A, IL-17A/F, IL-17F, and human IL-23 viasurface plasmon resonance. The association rate constant (k_(a)(M⁻¹s⁻¹)) is a value that reflects the rate of the antigen-antibodycomplex formation. The dissociation rate constant (k_(d) (s⁻¹)) is avalue that reflects the stability of this complex. By dividing thedissociation rate constant by the association rate constant(k_(d)/k_(a)) the equilibrium dissociation constant (K_(D) (M)) isobtained. This value describes the binding affinity of the interaction.Antibodies with similar K_(D) can have widely variable association anddissociation rate constants. Consequently, measuring both the k_(a) andk_(d) of antibodies helps to more uniquely describe the affinity of theantibody-antigen interaction.

Binding kinetics and affinity studies were performed on a BIACORE® T100system (GE Healthcare, Piscataway, N.J.). Methods for the BIACORE® T100were programmed using BIACORE® T100 Control Software, v 2.0. For theseexperiments, the monoclonal and bispecific antibodies were captured ontoa CM4 sensor chip via goat anti-human IgG Fc-gamma antibody (JacksonImmunoResearch, West Grove, Pa.). Binding experiments with the humanIL-17 molecules were performed at 25° C. in a buffer of 10 mM HEPES, 150mM NaCl, 3 mM EDTA, 0.05% Surfactant P20 (GE Healthcare), 1 mg/mL bovineserum albumin, pH 7.4. Binding experiments with the IL-23/IL-12Bheterodimer were performed at 25° C. in a buffer of 10 mM HEPES, 500 mMNaCl, 3 mM EDTA, 0.05% Surfactant P20 (Biacore), 1 mg/mL bovine serumalbumin, pH 7.4.

The capture antibody, goat anti-human IgG Fc-gamma, was diluted toconcentration of 20 μg/mL in 10 mM sodium acetate pH 5.0, and thencovalently immobilized to all four flow cells of a CM4 sensor chip usingamine coupling chemistry (EDC:NHS). After immobilization of theantibody, the remaining active sites on the flow cell were blocked with1 M ethanolamine. A capture antibody density of approximately 5000 RUwas obtained. The anti-human IL-23/IL-17A/F antibodies were capturedonto flow cell 2, 3, or 4 of the CM4 chip at a density ranging from60-150 RU. Capture of the test antibodies to the immobilized surface wasperformed at a flow rate of 10 μL/min. The BIACORE® instrument measuresthe mass of protein bound to the sensor chip surface, and thus, captureof the test antibody was verified for each cycle. Serial dilutions ofhuman recombinant IL-17A, IL-17A/F, or IL-17F (ZymoGenetics, ABristol-Myers Squibb Company, Seattle, Wash., USA) were prepared from100 nM-0.032 nM (1:5 serial dilutions), while serial dilutions of humanrecombinant IL-23 (ZymoGenetics, A Bristol-Myers Squibb Company,Seattle, Wash., USA) were prepared from 200 nM-0.064 nM (1:5 serialdilutions). The serial dilutions were injected over the surface andallowed to specifically bind to the test antibody captured on the sensorchip. Duplicate injections of each antigen concentration were performedwith an association time of 7 minutes and dissociation time of 15minutes. Kinetic binding studies were performed with a flow rate of 50μL/min. In between cycles, the flow cell was washed with 20 mMhydrochloric acid to regenerate the surface. This wash step removed boththe captured test antibody and any bound antigen from the immobilizedantibody surface. The test antibody was subsequently captured again inthe next cycle.

Data was compiled using the BIACORE® T100 Evaluation software (version2.0). Data was processed by subtracting reference flow cell and blankinjections. Baseline stability was assessed to ensure that theregeneration step provided a consistent binding surface throughout thesequence of injections. Duplicate injection curves were checked forreproducibility. Based on the binding of the bivalent IL-17 molecules toa bivalent antibody, the bivalent analyte binding interaction model wasdetermined to be appropriate for interactions with the IL-17 molecules.Based on the binding of the IL-23/IL-12B heterodimer to a bivalentantibody, the 1:1 binding interaction model was determined to beappropriate for interactions with the IL-23 molecule. The referencesubtracted binding curves were globally fit to the appropriate bindingmodel with a multiple Rmax and with the RI set to zero. The data fitwell to the binding models with good agreement between the experimentaland theoretical binding curves. The chi² and standard errors associatedthe fits were low. There was no trending in the residuals.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Biacore Activity

The results of the binding experiments with human IL-17A, IL-17A/F, andIL-17F are shown in Tables 10, 11, and 12, respectively. The results ofthe binding experiments with the human IL-23/IL-12B heterodimer areshown in Table 13.

Anti-Human IL-23/IL-17A/F Bispecific Antibodies Binding Affinity forIL-17A

TABLE 10 k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M)23/17bAb1 2.E+05 6.E−05 3.E−10 IgG1.1 SEQ ID NO: 28 SEQ ID NO: 1723/17bAb2 5.E+05 4.E−04 8.E−10 IgG1.1 SEQ ID NO: 18 SEQ ID NO: 1723/17bAb3 4.E+05 5.E−05 1.E−10 IgG4.1 SEQ ID NO: 74 SEQ ID NO: 1723/17bAb4 5.E+05 3.E−04 6.E−10 IgG4.1 SEQ ID NO: 29 SEQ ID NO: 1723/17taFab1 3.E+05 2.E−03 7.E−9  IgG1.1 SEQ ID NO: 76 SEQ ID NO: 78Anti-Human IL-23/IL-17A/F Bispecific Antibodies Binding Affinity forIL-17A/F

TABLE 11 k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M)23/17bAb1 2.E+05 9.E−05  4.E−10 IgG1.1 SEQ ID NO: 28 SEQ ID NO: 1723/17bAb2 4.E+05 7.E−04 2.E−9 IgG1.1 SEQ ID NO: 18 SEQ ID NO: 1723/17bAb3 2.E+05 2.E−04 1.E−9 IgG4.1 SEQ ID NO: 74 SEQ ID NO: 1723/17bAb4 3.E+05 1.E−03 3.E−9 IgG4.1 SEQ ID NO: 29 SEQ ID NO: 1723/17taFab1 1.E+05 5.E−04 5.E−9 IgG1.1 SEQ ID NO: 76 SEQ ID NO: 78Anti-Human IL-23/IL-17A/F Bispecific Antibodies Binding Affinity forIL-17F

TABLE 12 k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M)23/17bAb1 8.E+05 3.E−04 4.E−10 IgG1.1 SEQ ID NO: 28 SEQ ID NO: 1723/17bAb2 3.E+06 7.E−04 2.E−10 IgG1.1 SEQ ID NO: 18 SEQ ID NO: 1723/17bAb3 6.E+05 2.E−04 3.E−10 IgG4.1 SEQ ID NO: 74 SEQ ID NO: 1723/17bAb4 2.E+06 7.E−04 4.E−10 IgG4.1 SEQ ID NO: 29 SEQ ID NO: 1723/17taFab1 3.E+05 7.E−04 2.E−9  IgG1.1 SEQ ID NO: 76 SEQ ID NO: 78Anti-Human IL-23/IL-17A/F Bispecific Antibodies Binding Affinity forIL23/IL-12B

TABLE 13 Antibody or k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹)K_(D1) (M) 7B7Mab 3.E+05 2.E−04 7.E−10 SEQ ID NO: 68 SEQ ID NO: 1723/17bAb1 4.E+05 2.E−04 5.E−10 IgG1.1 SEQ ID NO: 28 SEQ ID NO: 1723/17bAb2 2.E+05 8.E−05 4.E−10 IgG1.1 SEQ ID NO: 18 SEQ ID NO: 1723/17bAb3 4.E+05 2.E−04 5.E−10 IgG4.1 SEQ ID NO: 74 SEQ ID NO: 1723/17bAb4 7.E+04 7.E−05 1.E−9  IgG4.1 SEQ ID NO: 29 SEQ ID NO: 1723/17taFab1 3.E+05 2.E−04 7.E−10 IgG1.1 SEQ ID NO: 76 SEQ ID NO: 78Simultaneous Co-Binding of IL-17A/F and IL-23 to the Anti-HumanIL-23/IL-17A/F Bispecific Antibodies Via Surface Plasmon Resonance(Biacore)

Anti-Human IL-23/IL-17A/F bispecific antibodies were evaluated viasurface plasmon resonance for ability to simultaneously co-bind bothIL-23 and IL-17A/F.

For co-binding experiments in the first orientation, the human IL-17molecules were covalently immobilized to flow cells 2-4 of a CM5 sensorchip using amine coupling chemistry (EDC:NHS). After immobilization, theremaining active sites on the flow cells were blocked with 1 Methanolamine. Human IL-17A, IL-17A/F, and IL-17F (ZymoGenetics, ABristol-Myers Squibb Company, Seattle, Wash., USA) were immobilized ontoflow cells 2, 3, or 4 respectively. The immobilization levels of thesemolecules ranged from 4500-5200 RU. Flow cell 1 was used as thereference surface. The bispecific antibodies were subsequently dilutedto either 25 or 50 μg/mL, flowed over the surface, and captured ontoflow cells 2-4 of the sensor chip. Following capture of the bispecificantibody, the IL-23/IL-12B heterodimer (ZymoGenetics, A Bristol-MyersSquibb Company, Seattle, Wash., USA) was diluted to 500 nM and flowedover the surface to demonstrate co-binding. Binding studies wereperformed with a flow rate of 10 μL/min, an association time of 10minutes, and a dissociation time of 5 minutes.

For co-binding experiments in the second orientation, a mouse anti-humanIL-12 (p40/p70) monoclonal antibody (BD Pharmingen, San Jose, Calif.)was covalently immobilized onto flow cells 1-4 of a CM5 sensor chipusing amine coupling chemistry (EDC:NHS). After immobilization, theremaining active sites on the flow cells were blocked with 1 Methanolamine. The human IL-23/IL-12B heterodimer (ZymoGenetics, ABristol-Myers Squibb Company, Seattle, Wash., USA) was diluted to 500 nMand captured onto flow cells 1-4 via the IL-12B subunit. The capturelevel of the IL-23/IL-12B was approximately 4000 RU. The bispecificantibodies were subsequently diluted to either 25 or 50 μg/mL, flowedover the surface, and captured via the human IL-23 subunit onto flowcells 2-4 of the sensor chip. Flow cell 1 was used as the referencesurface. Following capture of the bispecific antibody, human IL-17A,IL-17A/F, and IL-17F (ZymoGenetics, A Bristol-Myers Squibb Company,Seattle, Wash., USA) were diluted to 500 nM and flowed over the surfaceto demonstrate co-binding. Binding studies were performed with a flowrate of 10 μL/min, an association time of 10 minutes, and a dissociationtime of 5 minutes.

All binding experiments were performed at 25° C. in a buffer of 10 mMHEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20 (GE Healthcare), 1mg/mL bovine serum albumin, pH 7.4. Between cycles, the flow cell waswashed with 20 mM hydrochloric acid to regenerate the surface. This washstep removed both the captured test antibody and any bound antigen fromthe chip surface. Data was compiled using BIACORE® T100 Evaluationsoftware (version 2.0). Data was processed by subtracting reference flowcell and blank injections. Baseline stability was assessed to ensurethat the regeneration step provided a consistent binding surfacethroughout the sequence of injections.

Simultaneous Co-Binding of IL-17A/F and IL-23 to the Anti-HumanIL-23/IL-17A/F Bispecific Antibodies Via Surface Plasmon Resonance(Biacore) Results

Bispecific antibodies tested include 23/17bAb1 (SEQ ID NO:28 and SEQ IDNO:17), 23/17bAb2 (SEQ ID NO:18 and SEQ ID NO:17), 23/17bAb3 (SEQ IDNO:74 and SEQ ID NO:17), 23/17bAb4 (SEQ ID NO:29 and SEQ ID NO:17), and23/17taFab1 (SEQ ID NO:76 and SEQ ID NO:78). All bispecific antibodieswere able to simultaneously co-bind both human IL-23 and human IL-17A/F,demonstrating that both arms of the bispecific antibodies werefunctional.

Demonstration of IL-17A/F Specific Binding of the Anti-HumanIL-23/IL-17A/F Bispecific Antibodies Via Surface Plasmon Resonance(Biacore)

Anti-Human IL-23/IL-17A/F bispecific antibodies were evaluated viasurface plasmon resonance for lack of cross reactivity to human IL-17B,human IL-17C, human IL-17D, and human IL-17E (ZymoGenetics, ABristol-Myers Squibb Company, Seattle, Wash., USA).

Binding studies were performed on a BIACORE® T100 (GE Healthcare,Piscataway, N.J.). Methods were programmed using BIACORE® T100 ControlSoftware, v 2.0. Goat anti-human IgG Fc-gamma specific antibody (JacksonImmunoResearch, West Grove, Pa.) was covalently immobilized to flowcells 1-3 of a CM4 sensor chip using amine coupling chemistry (EDC:NHS).The purified bispecific antibodies were subsequently captured ontoeither flow cell 2 or flow cell 3 of the sensor chip at a density ofapproximately 150 RU. Flow cell 1 was used as the reference surface.

Human IL-17B, IL-17C, IL-17D, and IL-17E (ZymoGenetics, A Bristol-MyersSquibb Company, Seattle, Wash., USA) were injected over the capturedantibody surface (flow cell 2) and the reference flow cell (flow cell 1)at concentrations of 500, 100, 20, and 4 nM. As a positive control forthis set of experiments, human IL-23 (ZymoGenetics, A Bristol-MyersSquibb Company, Seattle, Wash., USA) was injected at concentrations of100, 20, 4 and 0.8 nM. Binding studies were performed with a flow rateof 50 μL/min, an association time of 5 minutes, and a dissociation timeof 5 minutes. All binding experiments were performed at 25° C. in abuffer of 10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Surfactant P20 (GEHealthcare), 1 mg/mL bovine serum albumin, pH 7.4. Between cycles, theflow cell was washed with 20 mM hydrochloric acid to regenerate thesurface. This wash step removed both the captured test antibody and anybound antigen from the chip surface. Data was compiled using BIACORE®T100 Evaluation software (version 2.0). Data was processed bysubtracting reference flow cell and blank injections. Baseline stabilitywas assessed to ensure that the regeneration step provided a consistentbinding surface throughout the sequence of injections.

Demonstration of IL-17A/F Specific Binding of the Anti-HumanIL-23/IL-17A/F Bispecific Antibodies Via Surface Plasmon Resonance(Biacore) Results

No binding of human IL-17B, IL-17C, IL-17D, or IL17E to the bispecificantibodies was observed. Bispecific antibodies tested include 23/17bAb1(SEQ ID NO:28 and SEQ ID NO:17), 23/17bAb2 (SEQ ID NO:18 and SEQ IDNO:17), 23/17bAb3 (SEQ ID NO:74 and SEQ ID NO:17), 23/17bAb4 (SEQ IDNO:29 and SEQ ID NO:17). In contrast, the IL-23 positive controldemonstrated a dose dependent binding that was consistent with theprevious studies.

Example 4 Anti-Human IL-23/17A/F bAbs Prevent Human IL-17A, F andAF-Mediated Increases in Serum Concentrations of Murine KC (CXCL1) inMice

IL-17A, F and AF are able to induce the production of a number ofdownstream factors that in turn play a role in host defense, but alsocontribute to disease pathology, especially when produced at abnormallyhigh levels or under chronic conditions. One of these downstreammediators is CXCL1 (also known as GRO-α in human, or KC in mice), achemokine that has important neutrophil chemoattractant activity andplays a role in inflammation. The ability of anti-human IL23/17A/Fbispecific antibodies (bAbs) to reduce IL-17A, F and AF-mediatedincreases in GRO-α in mice was evaluated in order to show that the bAbswould be efficacious against IL-17-induced activities in an in vivosetting and thus that the bAbs would be useful in treating humandiseases in which IL-17A, F or AF play a role. However, because thesebAbs do not cross react with mouse IL-17A, F or AF, it was necessary todeliver human (h) IL-17A, F or AF to mice to induce the production ofGRO-α (or in the case of mice, induce the production of KC, which is themurine analogue of GRO-α) which could then be neutralized in thepresence of the anti-human IL23/17A/F bAbs.

For these experiments, female BALB/c mice (age 7-9 wk) were used. Attime 18 hours, the mice received an intra-peritoneal (i.p.) injection ofeither the vehicle (PBS) or a dose of one of the anti-human IL-23/17A/FbAbs as shown in Table 14 and 15, in the left-hand column. At time 0,they received a subcutaneous (s.c.) injection of one of the followingrecombinant human proteins: 0.175 mg/kg hIL-17A, 0.9 mg/kg hIL-17F or0.5 mg/kg hIL-17AF. Control mice received a s.c. injection of thevehicle (PBS) instead of one of the hIL-17 proteins. Two hours later,the mice were bled via the retro-orbital sinus under isoflurane gasanesthesia, serum was collected following centrifugation of the blood,and the serum was then stored at 80° C. until analyzed for serum KCconcentrations using a commercial ELISA as per the manufacturer'sinstructions (Quantikine Mouse CXCL1/KC Immunoassay, R&D Systems, Inc.,Minneapolis, Minn.).

As shown in Table 14 and 15, mice treated with the bAbs showed adose-dependent increase in the inhibition of hIL-17A, F or AF-inducedserum KC (CXCL1) concentrations indicating that the bAbs wereefficacious in reducing the activities mediated by these IL-17 ligands.CXCL1 is just one example of a biological readout in response to IL-17A,F or AF; there are numerous other important downstream readouts thatalso play a role in diseases in which IL-17A, F or AF play a role thatcould be used as endpoint measurement.

TABLE 14 Percent Inhibition of Human IL-17A or F-mediated Increases inSerum Concentrations of Murine KC by i.p. bAbs, Relative to theConcentrations of Vehicle-Treated Mice (n = 3-4 per Group) % Inhibitionof % Inhibition of IL-17A-Mediated Serum IL-17F-Mediated Serum KC LevelsKC Levels Vehicle (PBS) 0 0  1 mg/kg bAb1 81 76  5 mg/kg bAb1 90 88 12mg/kg bAb1 100 97  1 mg/kg bAb2 91 68  5 mg/kg bAb2 93 83 12 mg/kg bAb284 90  1 mg/kg bAb3 78 95  5 mg/kg bAb3 94 89 12 mg/kg bAb3 93 90  1mg/kg bAb4 94 51  5 mg/kg bAb4 87 89 12 mg/kg bAb4 94 92

TABLE 15 Percent Inhibition of Human IL-17AF-mediated Increases in SerumConcentrations of Murine KC (pg/mL) by i.p. bAbs, Relative to theConcentrations of Vehicle-Treated Mice (n = 4 per Group) % Inhibition ofIL-17AF-Mediated Serum KC Levels Vehicle (PBS) 0 0.3 mg/kg bAb1 55  10mg/kg bAb1 100 0.3 mg/kg bAb2 40  10 mg/kg bAb2 90 0.3 mg/kg bAb3 70  10mg/kg bAb3 96 0.3 mg/kg bAb4 10  10 mg/kg bAb4 72

Example 5 Anti-Human IL-23/17A/F bAbs Prevent Human IL-23-MediatedIncreases in Serum Concentrations of Mouse IL-17AF and F in Mice

IL-23 is able to induce the differentiation of Th17 cells which in turn,can lead to the production of IL-17A, IL-17F and IL-17AF. Thesecytokines are implicated in a number of diseases and therapeutics thatcan inhibit IL-23 and IL-17A, F and AF would be efficacious in thetreatment of these diseases. The ability of anti-human IL23/17A/Fbispecific antibodies (bAbs) to reduce IL-23-mediated increases inIL-17A, F and AF in mice was evaluated in order to show that the bAbswould be efficacious against IL-23-induced activities in an in vivosetting, and thus that the bAbs would be useful in treating humandiseases in which IL-23 and Th17 cells play a role. However, becausethese bAbs do not cross react with mouse IL-23 it was necessary todeliver human (h) IL-23 to mice to induce the production of mouse IL-17F and AF which could then be neutralized in the presence of theanti-human IL23/17A/F bAbs. Concentrations of mouse IL-17A were too lowto accurately measure in the mouse serum but the trends were expected tobe similar as compared to the trends observed for serum IL-17F and AF.

For these experiments, female C57BL/6 mice (age 7-9 wk) were used. At10:30 am on day 1, they each received 5 micrograms of mouse (m) IL-2 viaan intra-peritoneal (i.p.) injection. At 8:30 am on day 2, the micereceived an i.p. injection of either the vehicle (PBS) or a dose of oneof the anti-human IL-23/17A/F bAbs as shown in Table 16, in theleft-hand column. At 11 am on day 2 the mice each received 5 microgramsof mIL-2 and 10 micrograms of hIL-23, and at 5:20 pm on day 2, the micereceived 10 micrograms each of mIL-2 and hIL-23 via i.p. injections. At9:30 am on day 3, each of the mice received another 5 micrograms ofmIL-2 and 10 micrograms of hIL-23 by i.p. injection. At 4:30 pm on day3, the mice were bled via the retro-orbital sinus under isoflurane gasanesthesia, serum was collected following centrifugation of the blood,and the serum stored at −80° C. until analyzed for serum concentrationsof mouse IL-17F and AF using ELISAs and luminex assays that specificallymeasured these components.

As shown in Table 16, mice treated with the bAbs showed a dose-dependentincrease in the inhibition of hIL-23 induced serum concentrations ofmouse 17F or AF indicating that the bAbs were efficacious in reducingthe activities mediated by hIL-23.

TABLE 16 Percent Inhibition of Human IL-23 Mediated Increases in SerumConcentrations of Mouse IL-17F or AF by i.p. bAbs, Relative to theConcentrations of Vehicle-Treated Mice (n = 3 per Group) % Inhibition ofIL- % Inhibition of IL- 23-Mediated Serum 23-Mediated Serum mIL-17FLevels mIL-17AF Levels Vehicle (PBS) 0 0  1 mg/kg bAb1 49 22  5 mg/kgbAb1 99 94 12 mg/kg bAb1 96 94  1 mg/kg bAb2 21 17  5 mg/kg bAb2 82 7112 mg/kg bAb2 67 97  1 mg/kg bAb3 0 45  5 mg/kg bAb3 38 91 12 mg/kg bAb365 95  1 mg/kg bAb4 0 62  5 mg/kg bAb4 27 74 12 mg/kg bAb4 49 97

Example 6 VCVFc Bispecific Antibodies

Construction and Expression of Mammalian VCVFc Bispecific Molecules

Whole genes were synthesized at GenScript (GenScript, Piscataway, N.J.,USA) and inserted into pTT5, an HEK293-6E transient expression vector(NCR Biotechnology Research Institute, Ottawa, ON, CAN) via restrictionenzyme cloning. Most constructs were expressed using the mod 2610 (SEQID NO:30) signal sequence. The VCVFc is a bispecific antibody whichcontains a whole antibody with a Fv unit of the second arm of thebispecific inserted between the Fab region and the hinge via a linker(for example, but not limited to, 10 mer G₄S for either chain, orRTVAAPS (SEQ ID NO:85) for the light chain and SSASTKGPS (SEQ ID NO:86)for the heavy chain). An illustration of a VCVFc bispecific antibody isshown in FIG. 5.

The HEK293-6E suspension cells were transfected with expressionconstructs using polyethylenimine reagent and cultivated in F17 medium(Invitrogen, Grand Island, N.Y., USA) with the addition of 5 mML-glutamine and 25 μg/mL G418. After 24 hours, 1/40th volume of 20%Tryptone NI (Organotechnie SAS, La Courneuve, F R) was added. Atapproximately 120 hours post transfection, conditioned media washarvested and passed through a 0.2 μm filter. Protein was purified fromthe filtered conditioned media using a combination of Mab Select SuReAffinity Chromatography (GE Healthcare, Piscataway, N.J., USA) andSUPERDEX® 200 Size Exclusion Chromatography (GE Healthcare, Piscataway,N.J., USA). Content was estimated by absorbance at UV-A280 nm andquality evaluated by analytical size exclusion high performance liquidchromatography, SDS PAGE, and western blot.

IL-23/IL-17A/F VCVFc Bispecific Antibodies Bioassay Activity;NIH/3T3/KZ170 NE-κB Luciferase Reporter Assay to Measure Human IL-17A,IL-17A/F, and IL-17F Activity by NE-κB Induction

The bioassay was performed as described in Example 1 hereinabove.

IL-23/IL-17A/F VCVFc Bispecific Antibodies Bioassay Activity;Baf3/huIL-23Rα/huIL-12Rβ1 Transfectants Phospho-STAT3 Assay to MeasureHuman IL-23 Activity by Phospho-STAT3 Induction

The bioassay was performed as described in Example 3 hereinabove.

PDGF-C/PDGF-D VCVFc Bispecific Antibodies Bioassay Activity; NormalHuman Lung Fibroblasts (NHLF) Proliferation Assay to Measure HumanPDGF-C and PDGF-D Mitogenic Activity

A primary normal human lung fibroblast cell line (NHLF, CC-2512, Lonza,Walkersville, Md.) was seeded at 1,000 cells/well in growth media (FGM-2BulletKit, Lonza, Walkersville, Md.) and incubated overnight at 37° C.,5% CO₂. The following day media was removed and serial dilutions ofrecombinant human PDGF-C and PDGF-D (ZymoGenetics) were made up in assaymedia (FBM plus 0.1% BSA, Lonza, Walkersville, Md.) and added to theplates containing the cells and incubated together at 37° C., 5% CO₂ for48 hours. Additionally the assay was used to measure neutralizationPDGF-C and PDGF-D activity. A sub maximal concentration of PDGF-C orPDGF-D was combined with serial dilutions of anti-human PDGF-C/D oranti-human PDGFRα/β VCVFc antibodies described herein in assay media andadded to the plates containing the cells and incubated together at 37°C., 5% CO₂ for 48 hours. Cells were pulsed with 1 μCi/well of Thymidine[Methyl-³H] (PerkinElmer, Waltham, Mass.) and incubated at 37° C., 5%CO₂ for an additional 24 hours. Following incubation mitogenic activitywas assessed by measuring the amount of ³H-Thymidine incorporation.Media was removed and cells trypsinized for 10 minutes at 37° C. beforebeing harvested on FilterMate harvester (Packard Instrument Co.,Meriden, Conn.) and read on TOPCOUNT® microplate scintillation counter(Packard Instrument Co., Meriden, Conn.) according to manufacturesinstructions. Increases in ³H-Thymidine incorporation were indicative ofa PDGF-C or PDGF-D receptor-ligand interaction. Decreases in³H-Thymidine incorporation were indicative of neutralization of thePDGF-C or PDGF-D receptor-ligand interaction. IC₅₀ (inhibitoryconcentration at 50 percent) values were calculated using GraphPad Prism4 software (GraphPad Software, Inc., San Diego Calif.) for eachPDGF-C/PDGF-D or PDGFRα/PDGFRβ VCVFc bispecific antibody.

IL-23/IL-17A/F VCVFc Bispecific Antibody Bioassay Activity;NIH/3T3/KZ170 NE-κB Luciferase Reporter Assay andBaf3/huIL-23Rα/huIL-12Rβ1 Transfectants Phospho-STAT3 Assay Results

Human IL-17A, IL-17A/F and IL-17F induce activation of the NF-κBluciferase reporter in a dose dependent manner with an EC₅₀concentration determined to be 0.15 nM for IL-17A, 0.5 nM for IL-17A/Fand 0.5 nM for IL-17F and IL-23 induces STAT3 phosphorylation in a dosedependent manner with an EC₅₀ concentration determined to be 0.02 nM.The IC₅₀ data for the anti-human IL-23/IL-17A/F VCVFc bispecificantibodies are shown below in Tables 17, 18 and 19.

IL-23/17A/F VCVFc Bispecific Antibody Table

TABLE 17 Heavy Chain Light Chain MVC# MVC# IL-17A IL-17A/F IL-17F IL-23Name SEQ ID NO: SEQ ID NO: IC₅₀ nM IC₅₀ nM IC₅₀ nM IC₅₀ nM 339-134 mAbMVC978 MVC717 3 0.9 0.4 Not Done IgG1.1 SEQ ID SEQ ID NO: 64 NO: 66IL23.6 (7B7) MVC1003 MVC1002 Not Done Not Done Not Done 0.2 mAb IgG1.1SEQ ID SEQ ID NO: 68 NO: 17 23/17VCV1 MVC1020 MVC1021 20 3 3 0.008IgG1.1 SEQ ID SEQ ID NO: 87 NO: 89 23/17VCV2 MVC1022 MVC1023 0.4 0.4 0.90.3 IgG1.1 SEQ ID SEQ ID NO: 91 NO: 93

TABLE 18 Heavy Chain Light Chain MVC# MVC# IL-17A IL-17A/F IL-17F IL-23Name SEQ ID NO: SEQ ID NO: IC₅₀ nM IC₅₀ nM IC₅₀ nM IC₅₀ nM 339-134 mAbMVC978 MVC717 1.2 0.23 0.28 Not Done IgG1.1 SEQ ID SEQ ID NO: 64 NO: 66IL23.6 (7B7) MVC1003 MVC1002 Not Done Not Done Not Done 0.0030 mAbIgG1.1 SEQ ID SEQ ID NO: 68 NO: 17 23/17VCV3 MVC1119 MVC1021 16 9.7 6.00.011 IgG4.1 SEQ ID SEQ ID NO: 95 NO: 89 23/17VCV4 MVC1120 MVC1023 0.200.34 0.20 0.47 IgG4.1 SEQ ID SEQ ID NO: 97 NO: 93 23/17VCV5 MVC1122MVC1121 15 8.7 7.0 0.0038 IgG1.1 SEQ ID SEQ ID NO: 99 NO: 101 23/17VCV6MVC1124 MVC1123 0.38 0.35 0.29 0.043 IgG1.1 SEQ ID SEQ ID NO: 103 NO:105

TABLE 19 Heavy Chain Light Chain MVC# MVC# IL-17A IL-17A/F IL-17F IL-23Name SEQ ID NO: SEQ ID NO: IC₅₀ nM IC₅₀ nM IC₅₀ nM IC₅₀ nM 339.15.3.6N/A N/A 9.8 0.34 0.32 Not mAb Done Hybridoma line lot E10915 IL23.4 mAbSEQ ID SEQ ID Not Done Not Done Not Done 0.029 IgG4.1-BDC NO: 107 NO:109 Lot PC-1413- 32 23/17VCV7 MVC1108 MVC1107 24 13 5.9 0.053 IgG1.1 SEQID SEQ ID NO: 111 NO: 113 23/17VCV8 MVC1110 MVC1109 2.4 0.34 0.31 2.6IgG1.1 SEQ ID SEQ ID NO: 115 NO: 117PDGF-C/PDGF-D and PDGFRα/PDGFβ VCVFc Bispecific Antibodies BioassayActivity; Normal Human Lung Fibroblasts (NHLF) Proliferation AssayResults

PDGF-C and PDGF-D induce proliferation of the NHLF cells in a dosedependent manner with a sub maximal concentration determined to be 0.1nM for PDGF-C and 6 nM for PDGF-D. Table 20 and Table 21 present IC₅₀data for the PDGF-C/PDGF-D or PDGFRα/PDGFRβ VCVFc bispecific antibodydescribed herein.

PDGF-C/PDGF-D VCVFc Bispecific Antibody Table

TABLE 20 Heavy Chain Light Chain MVC# MVC# PDGFC PDGFD Name SEQ ID NO:SEQ ID NO: IC₅₀ nM IC₅₀ nM PDGFC mAb N/A N/A  .083 Not Done HybridomaLot-E2826 PDGFD mAb N/A N/A Not Done 3.5 Hybridoma Lot-E4342 C/DVCV1MVC1112 MVC1111 0.090 20 IgG1.1 SEQ ID SEQ ID NO: 121 NO: 119PDGFRα/PDGFRβ VCVFc Bispecific Antibody Table

TABLE 21 Heavy Chain Light Chain PDGFC PDGFD MVC# MVC# % Inhi- % Inhi-Name SEQ ID NO: SEQ ID NO: bition bition PDGFRα mAb N/A N/A 100%   30%Hybridoma Lot-C5161 PDGFRβ mAb N/A N/A 50% 100% Hybridoma Lot-C8938α/βVCV2 MVC1118 MVC1117 70% 100% IgG1.1 SEQ ID NO: 123 SEQ ID NO: 125IL-23/IL-17A/F VCVFc Bispecific Antibodies Bioassay Activity; PrimaryHuman Fibroblast Assay to Measure Human IL-17A, IL-17A/F, and IL-17FActivity by IL-6 Induction

The bioassay was pertextured as described in Example 3 hereinabove.

IL-23/IL-17A/F VCVFc Bispecific Antibodies Bioassay Activity; PrimaryHuman Fibroblast Assay Results

Human IL-17A, IL-17A/F and IL-17F induce human IL-6 production in a dosedependent manner with an EC₅₀ concentration determined to be 0.08 nM forIL-17A, 25 nM for IL-17A/F and 25 nM for IL-17F. Anti-humanIL-23/IL-17A/F VCVFc bispecific antibody 23/17VCV2 (SEQ ID NO:91 and SEQID NO:93). Table 22 presents example IC₅₀ data for the IL-23/IL-17A/FVCVFc bispecific antibody described herein.

TABLE 22 23/17VCV2 339-134 IL23.6(7B7) IgG1.1 mAbIgG1.1 mAbIgG1.1 SEQ IDNO: 91 SEQ ID NO: 64 SEQ ID NO: 68 Profile SEQ ID NO: 93 SEQ ID NO: 66SEQ ID NO: 17 Cellular Potency IL-17A 0.3 nM  2 nM Not Done Hu. primaryEC₅₀ = 0.08 nM fibroblast cells IL-17AF  26 nM 22 nM Not Done (HFFF)EC₅₀ = 25 nM IC₅₀ IL-17F  25 nM 23 nM Not Done EC₅₀ = 25 nM Cellularpotency IL-23 0.4 nM Not Done 0.02 nM Primary T cell EC₅₀ = 0.02 nMassay IC₅₀IL-23/IL-17A/F VCVFc Bispecific Antibodies Co-Binding Activity; PrimaryHuman Fibroblast Assay to Measure the Inhibition of Human IL-17A,IL-7A/F, or IL-F while Simultaneously Bound to Human IL-23. The PrimaryHuman T Cell Phospho-STAT3 Assay to Measure the Inhibition of HumanIL-23 while Simultaneously Bound to Human IL-17A, IL-7A/F, or IL-17F.

The primary human fibroblast assay was run in the presence of excessamounts of IL-23 at 30 nM. The primary human T cell phospho-STAT3 assaywas run in the presence of excess amounts of IL-17A, IL-17A/F, andIL-17F at 30 nM.

IL-23/IL-17A/F VCVFc Bispecific Antibodies Bioassay Co-Binding Results

Bispecific antibody 23/17VCV2 (SEQ ID NO:91 and SEQ ID NO:93) whenexamined in the presence of human IL-23 did not interfere with humanIL-17A, IL-17A/F, IL-17F inhibition. Bispecific antibody 23/17VCV2 whenexamined in the presence of human IL-17A, IL-17A/F, IL-17F did notinterfere with human IL-23 inhibition.

Measurement of Binding Affinities of IL-23/IL-17A/F VCVFc BispecificAntibodies to Human IL-17A, IL-17A/F, IL-17F, and Human IL-23 ViaSurface Plasmon Resonance (Biacore)

Binding activities were determined as described in Example 3hereinabove.

IL-23/IL-17A/F VCVFc Bispecific Antibodies Biacore Activity

The results of the binding experiments with human IL-17A, IL-17A/F, andIL-17F are shown in Tables 23, 24, and 25, respectively. The results ofthe binding experiments with the human IL-23/IL-12B heterodimer areshown in Table 26.

IL-23/IL-17A/F VCVFc Bispecific Antibodies Binding Affinity for IL-17A

TABLE 23 k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M)K_(D1) (nM) 339-134 mAb  2.E+06  3.E−03 1.E−9 1.0 IgG1.1 SEQ ID NO: 64SEQ ID NO: 66 23/17VCV2 3.8E+06 3.4E−03 8.9E−10 0.9 IgG1.1 SEQ ID NO: 91SEQ ID NO: 93 23/17VCV4 5.4E+06 5.4E+03 1.0E+09 1.0 IgG4.1 SEQ ID NO: 97SEQ ID NO: 93 23/17VCV6 4.0E+06 4.7E+03 1.2E+09 1.2 IgG1.1 SEQ ID NO:103 SEQ ID NO: 105IL-23/IL-17A/F VCVFc Bispecific Antibodies Binding Affinity for IL-17A/F

TABLE 24 k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M)K_(D1) (nM) 339-134 mAb  2.E+06  5.E−04  2.E−10 0.2 IgG1.1 SEQ ID NO: 64SEQ ID NO: 66 23/17VCV2 1.8E+06 7.1E−04 3.9E−10 0.4 IgG1.1 SEQ ID NO: 91SEQ ID NO: 93 23/17VCV4 1.5E+06 7.7E+04 5.1E+10 0.5 IgG4.1 SEQ ID NO: 97SEQ ID NO: 93 23/17VCV6 1.2E+06 7.9E+04 6.6E+10 0.7 IgG1.1 SEQ ID NO:103 SEQ ID NO: 105IL-23/IL-17A/F VCVFc Bispecific Antibodies Binding Affinity for IL-17F

TABLE 25 k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹) K_(D1) (M)K_(D1) (nM) 339-134 mAb  2.E+06  2.E−04  1.E−10 0.1 IgG1.1 SEQ ID NO: 64SEQ ID NO: 66 23/17VCV2 2.4E+06 5.1E−04 2.1E−10 0.2 IgG1.1 SEQ ID NO: 91SEQ ID NO: 93 23/17VCV4 2.2E+06 3.5E+04 1.6E+10 0.2 IgG4.1 SEQ ID NO: 97SEQ ID NO: 93 23/17VCV6 3.4E+06 1.2E+04 3.5E+11 0.04 IgG1.1 SEQ ID NO:103 SEQ ID NO: 105IL-23/IL-17A/F VCVFc Bispecific Antibodies Binding Affinity forIL23/IL-12B

TABLE 26 Antibody or k_(a1) k_(d1) Bispecific Antibody (M⁻¹s⁻¹) (s⁻¹)K_(D1) (M) K_(D1) (nM) 7B7Mab  3.E+05  2.E−04  7.E−10 0.7 SEQ ID NO: 68SEQ ID NO: 17 23/17VCV2 4.7E+04 2.1E−04 4.5E−09 4.5 IgG1.1 SEQ ID NO: 91SEQ ID NO: 93 23/17VCV4 No No No No IgG4.1 Binding Binding BindingBinding SEQ ID NO: 97 SEQ ID NO: 93 23/17VCV6 5.6E+04 1.1E+04 2.0E+092.0 IgG1.1 SEQ ID NO: 103 SEQ ID NO: 105Simultaneous Co-Binding of IL-17A/F and IL-23 to the IL-23/IL-17A/FVCVFc Bispecific Antibodies Via Surface Plasmon Resonance (Biacore)

This assay was performed as described in Example 3 hereinabove.

Simultaneous Co-Binding of IL-17A/F and IL-23 to the IL-23/IL-17A/FVCVFc Bispecific Antibodies Via Surface Plasmon Resonance (Biacore)Results

Bispecific antibody 23/17VCV2 (SEQ ID NO:91 and SEQ ID NO:93) was ableto simultaneously co-bind both human IL-23 and human IL-17A/F,demonstrating that both arms of the bispecific antibodies werefunctional.

Example 7 IL-23p19 Epitope Mapping

The analysis described in this Example 7 aims to identify the epitopicresidues on IL-23p19 for which the IL-23p19 antibody (7B7 antibody orMab, 7B7 Fab and biAb3, all of which have a heavy chain variable domainas shown in SEQ ID NO:7 and light chain variable domain as shown in SEQID NO:9) binds. Fab 7B7, 7B7 antibody and biAb3 have all been used inthe binding studies at various stages because they are interchangeableas far as their epitope on IL-23p19.

Proteolytic Digest and Peptide Data on Epitope

Mass spectrometry epitope sequence analysis of the IL-23p19 antibody wasbased on both epitope extraction and epitope excision methods. (Parkeret al., “MALDI/MS-based epitope mapping of antigens bound to immobilizedantibodies”, Mol. Biotechnol., 20(1):49-62 (January 2002)). In bothcases the IL-23p19 antibody was directly immobilized via primary aminesof the antibody on surface-activated beads at an average density of 2 mgmAb per 1 ml bed volume. Peptides from IL-23 his-tag antigen weregenerated with or without reduction and alkylation. Reduction of theantigen IL-23 was performed by incubating with 50 mM dithiothreitol inPBS and 4M guanidine HCl for 1 hour at 37° C. This was followed byalkylation with 100 mM iodoacetamide for 30 minutes at room temperature.Reduced and alkylated IL-23 was dialyzed against PBS overnight prior tofragmentation. For epitope extraction, antigen peptides were generatedby proteolytic digestion with the endoproteinases trypsin, chymotrypsin,lys-C, arg-C, asp-N and or glu-C with an enzyme to antigen ratio of upto 2% (w/w). Incubations were performed at 37° C. with incubation timesranging from 2 hours to overnight. The resulting peptides were mixedwith antibody resin at room temperature for 30 minutes. This resin wasthen washed three times to remove any non-specifically bound peptides.All digestion, incubation, and wash steps were performed in PBS pH 7.The same protocol was followed for epitope excision except that theintact antigen was incubated with the antibody for 30 minutes at roomtemperature prior to enzymatic digestion. In both methods antibody boundpeptides were eluted and analyzed on ESI-MS.

These data indicate that IL-23p19 antibody has a discontinuous epitopecomprised of three peptide regions in IL-23p19. Synthetic peptides weregenerated to further examine these three peptide regions, and theirbinding was tested and analyzed by both ELISA and mass spectrometry.Based on these observations, it is suggested that the following peptidesrepresent the sequences of the IL-23p19 antibody epitope:

Peptide 1: WQRLLLRFKILR (residues 156-167 of SEQ ID NO: 6)Peptide 2: SAHPLVGHMDLR (residues 46-57 of SEQ ID NO: 6)Peptide 3: IHQGLIFYEKLLGSDIFTGEPSLLP (residues 93-117 of SEQ ID NO: 6).IL-23 Epitope Mapping by HDX-MS

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) method probesprotein conformation and conformational dynamics in solution bymonitoring the rate and extent of deuterium exchange of backbone amidehydrogen atoms. The level of HDX depends on the solvent accessibility ofbackbone amide hydrogen atoms and the conformation of the protein. Themass increase of the protein upon HDX can be precisely measured by MS.When this technique is paired with enzymatic digestion, structuralfeatures at the peptide level can be resolved, enabling differentiationof surface exposed peptides from those folded inside. Typically, thedeuterium labeling and subsequent quenching experiments are performed,followed by online pepsin digestion, peptide separation, and MSanalysis. Prior to epitope mapping of BMS-986113 in IL-23 by HDX-MS,non-deuteriated experiments were performed to generate a list of commonpeptic peptides for IL-23 (4.4 mg/mL) and IL-23/BMS-986113 (1:1 molarratio, 4.4 mg/mL & 3.36 mg/mL), achieving a sequence coverage of 97% forIL-23. In this experiment, 10 mM phosphate buffer (pH 7.0) was usedduring the labeling step, followed by adding quenching buffer (200 mMphosphate buffer with 1.5M GdnCl and 0.5M TCEP, pH 2.5, 1:1, v/v). Forepitope mapping experiments, 5 μL of each sample (IL-23 orIL-23/BMS-986113 (1:1 molar ratio)) was mixed with 65 μL HDX labelingbuffer (10 mM phosphate buffer in D₂O, pD 7.0) to start the labelingreactions at room temperature (˜25° C.). The reactions were carried outfor different periods of time: 20 sec, 1 min, 10 min, 60 min and 240min. By the end of each labeling reaction period, the reaction wasquenched by adding quenching buffer (1:1, v/v) and the quenched samplewas injected into Waters HDX-MS system for analysis. The observed commonpeptic peptides were monitored for their deuterium uptake levels in theabsence/presence of BMS-986113. The same protocol was followed forepitope mapping of anti-IL-23 7B7 Fab (4.91 mg/mL) in IL-23 by HDX-MS.

Epitope mapping of anti-IL-23 7B7 Fab in complex with IL-23 and biAb3with IL-23 indicate that biAb3 has a discontinuous epitope comprised offive peptide regions in IL-23p19. Based on relative deuterium uptakelevels, five peptide regions can be ranked as region 1>2>3>4>5 withregion 1 having the most significant changes in deuterium uptakes andregion 5 having the least significant changes in deuterium uptakes. Thefive peptide regions on IL-23p19 as determined by HDX-MS for theIL-23p19 antibody were determined as follows:

Region 1: PDSPVGQL (residues 117-124 of SEQ ID NO: 6);Region 2: IFTGEPSLL (residues 108-116 of SEQ ID NO: 6);Region 3: KILRSLQAF (residues 164-172 of SEQ ID NO: 6);Region 4: QQLSQKLCTLAWSAHPLVGHMD (residues 34-55 of SEQ ID NO: 6); andRegion 5: CLQRIHQGLIFYEKLLG (residues 89-105 of SEQ ID NO: 6).Computational Epitope Prediction and Design of Alanine Shave Mutants

Alanine shave mutagenesis is a strategy for mutating multiple residuesin the same construct to alanine to remove the amino acid side chains inepitope of binding (Wells, J. A., “Systemic mutational analyses ofprotein-protein interfaces”, Enzym., 202:390-411 (1991)). Multiplesources of information on the involvement of residues in a potentialepitope with the 7B7 Fab and biAb3 were combined to produce a targetedlist of regional alanine shave mutants. The residues contained in theoverlapping regions between both the HDX (see above in this Example 7)and the proteolytic digest peptide mapping (see above in this Example 7)were mapped onto the sequence of the IL-23p19 domain and three linearregions of common residues were identified as Regions A, B and C. RegionA corresponds to amino acid residues 33-59 of SEQ ID NO:6. Region Bcorresponds to amino acid residues 89-125 of SEQ ID NO:6. Region Ccorresponds to amino acid residues 144-173 of SEQ ID NO:6. In order tocalculate the residues whose side chains are exposed (solvent accessiblesurface area, SASA) and would therefore be located on the proteinsurface of the p19 domain of IL-23, an in-house structure of the IL-23heterodimer was used. For each residue in the p19 domain of IL-23 theratio of accessible surface to the standard exposed surface for theamino acid type was calculated and residues were grouped into bins.Residues were placed in accessibility bins as follows: <30%, 30-40%,40-50%, 50-60%, 60-70%, 70-80%, >90% exposed. The standard residueaccessibilities for each amino acid type were calculated in the extendedtripeptide Gly-X-Gly. The second calculation performed was ODA (OptimalDocking Area) which is useful for predicting likely protein-proteininteraction surfaces. The method identifies optimal surface patches withthe lowest docking desolvation energy. The ODA was calculated for thep19 domain of IL-23 and these results used to prioritize residues formutagenesis.

Residues in these three regions (A, B, C) were prioritized based upon ahigh score in both the ODA and SASA calculations and also weighted basedon the extent of hydrogen-deuterium exchange relative to the uncomplexedIL-23 (peptide #1>#2>#3>#4>#5). Regions with non-identity to mouse werecloned as mouse swap mutants, instead of alanine shave mutants, whichdid not show an impact on binding in small scale testing. With theexception of M7 which contains a linear sequence of residues in anextended loop, residues were then combined into non-linear epitopesbased on mapping them to the X-ray crystallographic structure of IL-23(M5, M6, M8). Additional backup mutants were generated with thesub-epitopes of predominantly linear residues (M9, M10, M11). Thealanine shave mutants designed by this method are shown below in Table27.

TABLE 27 Alanine Shave Mutants of IL-23p19 Region IL-23p19 (SEQ ID NO:6) Name Mutated Residues Mutated to Alanine M5 A and B H53A, M54A,E112A, L116A and D118A M6 A and C T42A, W45A, H48A, F163A and Q170A M7 CW142A, E143A, T144A, Q145A and Q146A M8 A, B and C H53A, E112A, Q154Aand W156A M9 B L116A, D118A and Q123A M10 A H53A, M54A, D55A and F163AM11 C W142A, T144A and Q146ACloning, Expression and Purification of IL-23 Epitope Mapping AlanineMutants

Non-tagged wild-type IL-23 p40 subunit entry vector construct wasgenerated by PCR and the fidelity of the PCR fragment was confirmed bysequencing. The transient expression construct was generated by GatewayLR recombination and sequence confirmed. The His-tagged wild-type IL-23p19 subunit construct and all mutant constructs were generated by PCRand cloned into the transient expression vector directly. The fidelityof all PCR fragments was confirmed by sequencing. To generate thewild-type control, non-tagged wild-type IL-23 P40 subunit wasco-expressed with the His-tagged wild-type P19 subunit transiently inHEK293-6E cells at 4 L scale for IL-23 complex purification. Briefly,HEK293-6E cells at 1×10⁶ cells/ml were transfected with expressionplasmids/PEI complex at the ratio of 0.5 (p19)/0.5 (p40)/1.5 (PEI).Tryptone N1 feed was added 24 hours later and cells harvested on 120hours post transfection. The conditioned media was filtered with 0.2 μMfilters. Seven His-tagged IL-23 p19 mutant constructs wereco-transfected with the non-tagged wild-type IL-23 p40 subunit at the 30ml scale following the same transfection protocol described above. Theconditioned media were transferred for analysis and the expression ofall mutants was confirmed by anti-His Western-blot. Based on preliminarybinding results, mutants M5, M7, M9, and M10 were selected and scaled upat 2 L scale with the same transfection protocol. The wild-type was alsoscaled up at 2 L. The scale-ups of wild-type and mutants of IL-23 at 2liters of HEK cells were harvested and the supernatants wereconcentrated and buffer exchanged to PBS by tangential flow filtrationwith a 10 kDa membrane. The proteins were then purified by immobilizednickel affinity chromatography. The wild-type was eluted with 40 mMimidazole and then buffer exchanged by desalting gel filtrationchromatography to PBS (5.6 mM Na₂HPO₄, 1.1 mM KH₂PO₄, 154 mM NaCl, pH7.4). The purity of the wild-type was determined by SDS-PAGE to be >95%.The mutants were washed with 40 mM imidazole followed by elution at 500mM Imidazole. The elution pools were then buffer exchanged by dialysisto PBS (7 mM Na₂HPO₄, 3 mM NaH₂PO₄, 130 mM NaCl, pH 7.1). The purity ofthe mutants was determined by SDS-PAGE to be >95%. Single alaninemutants of his-tagged IL-23p19 at key residues identified by alanineshave mutagenesis were generated by gene synthesis and then cloned intothe transient transfection vector. Expression and purification weresimilar to the alanine shave mutants with the exception of M35A whichwas an affinity purified.

Biacore Binding Analysis of IL-23 Mutants to the IL-23p19 Antibody

The binding of the 30 mL small scale expression of all seven (7) alaninemutants (see Table 27) was measured by surface Plasmon resonance (SPR,Biacore)) on a BIACORE® T100 in PBST (7 mM Na₂HPO₄, 3 mM NaH₂PO₄, 130 mMNaCl, 0.05% Tween 20, pH 7.4) at 25° C. The relevant antibodies andreceptors were captured at a level of about 60 RUs by protein Aimmobilized at 2000 RUs on a CM5 sensor chip. In addition to the biAb3,Merck's IL-23 p19 mAb (7G10) and STELARA® (IL-12/IL-23 p40 mAb) wereused as controls for domain binding. In addition, the commercialreceptors for IL-23 were used as controls: hIL-23R-Fc and hIL-12Rβ1-Fc(both from R&D Systems). The supernatants were diluted 1:5 into PBST andinjected at 30 μL/min over the mAb or receptor surface for 3 minutesand, after a dissociation time, regenerated with 10 mM Glycine, pH 2.0.Binding to a reference surface of Protein A without any capturedantibody was subtracted from all specific binding curves beforeanalysis. The results shown in FIG. 9 show the response 10 secondsbefore the end of the injection.

The Biacore results demonstrate that the IL-23 alanine shave mutantsmaintain binding to the p40 specific antibody and the p19 specific IL-23receptor (except for M8 which potentially describes the receptor bindingsite). Binding of another IL-23p19 mAb and IL-12Rβ1-Fc was alsoperformed and is consistent with the results of FIG. 9. The M5 mutantshows a dramatic loss of binding to 7B7 Mab while M9 & M10 show partialloss of binding to 7B7 Mab (see FIG. 10). M7 does not show any impact tobinding to the antibody or controls. Therefore, these four mutants werechosen for scale-up and purification, the three mutants that losebinding to the IL-23p19 antibody (7B7 antibody or Mab, 7B7 Fab andbiAb3, all of which have a heavy chain variable domain as shown in SEQID NO:7 and light chain variable domain as shown in SEQ ID NO:9) and theM7 as a control.

Biacore analysis of the purified mutants used the same assay format asthe supernatants except that the wild-type and mutant IL-23 was dilutedto 25 nM and serially titrated 1:2 down to 1.5 nM. All results obtainedwith the purified IL-23 mutants confirmed the data obtained with theexpression supernatants. The data were fit to a 1:1 Langmuir bindingmodel to determine the Kd values shown in FIG. 10 and Table 28. The 1-2RU of reference subtracted binding observed for M5 binding to theIL-23p19 antibody (7B7 antibody or Mab, 7B7 Fab and biAb3, all of whichhave a heavy chain variable domain as shown in SEQ ID NO:7 and lightchain variable domain as shown in SEQ ID NO:9) was simulated using theBIAsimulation software 2.1 using the average Rmax determined fromkinetic analysis of M9 & M10. The affinity was estimated to be ≧330 μMwith a 1500 fold weaker Kd than wild-type as shown in Table 28.

TABLE 28 Biacore Kinetic Analysis of IL-23 Alanine Mutants BindingIL-23p19 Antibody 7B7 mab Merck Kd-shift BiAb3 STELARA ® 7G10 (from ΔΔGΔΔG ΔΔG ΔΔG Variant Kd (nM) WT) (kcal/mole) (kcal/mole) (kcal/mole)(kcal/mole) WT 0.14 NA NA NA NA NA M5 ≧300 1500 4.6 3.8 0.1 1.9 M7 0.4 30.5 NM 0.1 0.2 M9 25 140 2.9 2.3 0.1 0.2 M10 43 230 3.2 2.3 0.3 0.4

Biacore analysis of single alanine mutants was performed to confirm thenon-linear epitope demonstrated by the M5, M9, and M10 alanine shavemutants. Most single alanine mutants in the three linear regions A, B,and C showed no change in binding to the 7B7 mAb or biAb3. Only three ofthe fourteen single alanine mutant of IL-23 tested showed a significantdecrease in binding affinity greater than 1 kcal/mole. The affinity andthe ΔΔG values for the key residues overlapping between alanine shavemutants M5, M9, and M10 shown in Table 29 and demonstrate that one majorresidue in linear region B and two residues in linear region Acontribute predominantly to the binding energy of the IL-23p19antibody-IL-23 complex.

TABLE 29 Biacore Kinetic Analysis of IL-23 Single Alanine MutantsBinding IL-23p19 Antibody 7B7 mab Merck Kd-shift BiAb3 STELARA ® 7G10(from ΔΔG ΔΔG ΔΔG ΔΔG Variant Kd (nM) WT) (kcal/mole) (kcal/mole)(kcal/mole) (kcal/mole) WT 0.14 NA NA NA NA NA His53 0.2 0 0.2 0.2 0.3 0(region A) Met 54 6.8 48 2.3 2.2 0.3 1.0 (region A) Asp55 9.6 68 2.5 2.30.5 0.7 (region A) Glu112 0.7 5 0.9 1.2 0.3 1.7 (region B) Leu116 46 3303.4 3.0 0.5 0.2 (region B) Asp118 0.1 0 0 −0.4 −0.3 −0.3 (region B)IL-23 Induced STAT3 Phosphorylation in BaF3/huIL-23Rα/huIL-12Rβ1Transfectants

A murine bone marrow derived cell line (BaF3) was stably transfectedwith human IL-23Rα and human IL-12Rβ1 full length receptors and cloned.IL-23 induction of phosphorylation of STAT3 was monitored by ELISA forIL-23 Alanine shave mutants. BaF3/huIL-23Rα/huIL-12Rβ1 (clone 6) cellswere washed three times with assay media before being plated at 50,000cells per well in 96-well round-bottom tissue culture plates.BaF3/huIL-23Rα/huIL-12Rβ1 cells respond to IL-23 in a dose dependantmanner by phosphorylation of STAT3. To assess antibody inhibition ofIL-23 signaling, an EC₅₀ concentration of 20 pM IL-23 was premixed withthree-fold serial dilutions of each antibody from 33 nM to 0.56 pM andincubated at 37° C. for 15 minutes in assay media prior to addition tothe cells. Following pre-incubation, treatments were added in duplicateto plates containing cells and incubated at 37° C. for 15 minutes tostimulate phosphorylation of STAT3. Stimulation was stopped with theaddition of ice-cold wash buffer and cells lysed according tomanufacturer's instructions (Bio-Rad Laboratories Cell Lysis kit, Cat#171-304012). Phosphorylated STAT3 levels were determined by ELISA(Bio-Rad Laboratories Phospho-STAT3^((Tyr705)) kit, Cat #171-V22552)according to manufacturer's instructions. Data was analyzed and IC₅₀values were calculated using GraphPad Prism 4 software. All the IL-23Alanine shave mutants and all the IL-23 Alanine single mutants areactive and equal potent as wt IL-23 (untagged and tagged) at inducingpSTAT3 activity on BaF3/huIL-23Rα/huIL-12Rβ1 transfectants (Table 30).Results for biAb3, STELARA® (IL-12/IL-23 p40 mAb), and Merck's IL-23p19antibody (7G10) inhibition of IL-23 Alanine shave mutant induced pSTAT3are shown in Table 31. biAb3 neutralizes the biological activity of wtIL-23 and IL-23 M7 Alanine shave mutant with equal potency, M9 and M10with reduced potency, and does not neutralize the biological activity ofM5 on BaF3/huIL-23Rα/huIL-12Rβ1 transfectants. STELARA® IL-12 p40 mAbneutralizes the biological activity of wt IL-23 and all the IL-23Alanine shave mutants with equal potency on BaF3/huIL-23Rα/huIL-12Rβ1transfectants. Positive control Merck IL-23 p19 mAb (7G10) neutralizesthe biological activity of wt IL-23, M7, M9 and M10 Alanine shavemutants with equal potency and does not neutralize the biologicalactivity of M5 on BaF3/huIL-23Rα/huIL-12Rβ1 transfectants.

TABLE 30 EC₅₀ values for IL-23 Alanine shave mutants and IC₅₀ Values forbiAb3, STELARA ® (IL-12/IL-23 p40 mAb), and Merck's IL-23p19 antibody(7G10) inhibition of IL-23 Alanine shave mutants induced pSTAT3 inBaF3/huIL-23Rα/huIL-12Rβ1 transfectants wt human IL-23 IL-23 IL-23 IL-23Antibody IL-23 M5 M7 M9 M10 None 21 pM 26 pM 21 pM 33 pM 19 pM (EC₅₀)biAb3 19 pM NA 17 pM 2400 pM  5300 pM  STE- 79 pM 62 pM 59 pM 67 pM 71pM LARA ® Merck 380 pM  NA 310 pM  260 pM  350 pM  7G10

Single IL-23p19 mutations (H53A, M54A and D55A) were constructed andIC₅₀ Values for 7B7, STELARA® (IL-12/IL-23p40 mAb), and Merck's IL-23p19antibody (7G10) (Table 31) to inhibit the single IL-23p19 mutatedpolypeptides to induce pSTAT3 in BaF3/huIL-23Rα/huIL-12Rβ1 transfectantswas determined. The 7B7 mAb neutralizes the biological activity of IL-23Alanine single mutants H53A, E112A, and D118A with equal potency, M54Aand D55A mutants with significantly reduced potency, and does notneutralize the biological activity of L116A mutant compared to wt IL-23on BaF3/huIL-23Rα/huIL-12Rβ1 transfectants (Table 31). STELARA® IL-12p40 mAb neutralizes the biological activity of all the IL-23 Alaninesingle mutants with equal potency compared to wt IL-23 onBaF3/huIL-23Rα/huIL-12Rβ1 transfectants. Merck IL-23 p19 mAb neutralizesthe biological activity of all the IL-23 Alanine single mutants withequal potency compared to wt IL-23 on BaF3/huIL-23Rα/huIL-12Rβ1transfectants, except for IL-23 E112A mutant which it does notneutralize.

TABLE 31 IC₅₀ Values for 7B7, STELARA ® (IL-12 /IL-23p40 mAb), andMerck's IL-23p19 Antibody (7G10) to Inhibit IL-23p19 Single Mutations(H53A, M54A and D55A) to Induce pSTAT3 in BaF3/huIL-23Rα/huIL-12Rβ1Transfectants IL-23 IL-23 IL-23 IL-23 IL-23 IL-23 H53A M54A D55A E112AL116A D118A IL-23 wt IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ IC₅₀ Antibody IC₅₀ (nM)(nM) (nM) (nM) (nM) (nM) (nM) 7B7/biAb3 0.020 0.015 0.71 1.2 0.025 >30.012 STELARA ® 0.067 0.078 0.081 0.077 0.033 0.054 0.052 p40 Merck 7G100.18 0.19 0.27 0.18 >3 0.24 0.22

Taking all these results together, 7B7 mAb inhibition of IL-23 requiresamino acids residues M54, D55, and L116, but does not require amino acidresidues H53, E112, or D118. STELARA® and Merck antibodies were ableinhibit the IL-23 Alanine single mutants M54A, D55A and L116A with noloss in activity, suggesting the loss in activity seen with IL-23 M54,D55 and L116 Alanine mutants are specific to the 7B7 mAb and the biAb3.

Oligomeric State Analysis of IL-23 Mutants and 7B7 Fab Complexes

To confirm the oligomeric state of the heterodimeric IL-23 mutants andtheir ability to complex with the 7B7 Fab, the proteins were studied byanalytical size-exclusion chromatography (SEC-MALS) separation using anAGILENT® 1100 series HPLC fitted with a diode-array absorbance detector,on a Shodex Protein KW-803 column in buffer (0.1 um filtered) containing200 mM K₂PO₄ (pH 6.8 with HCl), 150 mM NaCl, and 0.02% sodium azide, ata flow rate of 0.5 mL/min. A Wyatt Technology MINIDAWN™ TREOS® laserlight scattering instrument was plumbed downstream from the HPLC,followed by a Wyatt OPTILAB® T-REX™ differential refractometer. Sixty(60) μg of each IL-23 sample was injected after filtering at aconcentration of 10.3 μM. For complex formation, 60 μg of the 7B7 Fabwas premixed with a 6% molar excess and incubated with the IL-23 proteinat a concentration of 10.9 μM at room temperature for at 3-6 hoursbefore chromatographic separation. Particulates were removed fromprotein samples with a spin filter (NANOSEP® MF, 0.2 μm, PallCorporation) prior to injection. Data were analyzed with ASTRA® 6(Wyatt) and Chemstation (Agilent). All mutants were mostly monomeric,similar to the wild-type. The M5 mutant did not show significant complexformation after pre-incubation with the Fab and eluted close to wherethe M5 IL-23 alone elutes demonstrating that little if any complex wasformed in the 10 μM concentration range of this experiment. M7 complexedand eluted similar to the wild-type. The M9 and M10 mutants form complexat these concentrations, but the mass was somewhat less than that of thewild-type and the retention time was slightly later than the wild-typeand M7, suggesting that their affinity for 7B7 was weaker than thewild-type and M7. SEC-MALS analysis of the 14 single alanine mutantsshowed that all were mostly monomeric, similar to the wild-type, andonly the L116A mutant showed a late-shifted elution time and a slightreduction in the expected mass of the complex suggesting that theaffinity of the IL-23 for the 7B7 Fab was reduced. These results areconsistent with the Biacore shift in Kd for these L116A. The effect ofthe reduced affinity of the complex of D54A and M55A with the Fab wasnot able to be resolved under the conditions of this assay.

Differential Scanning Calorimetry of IL-23 Mutants

The thermal stability profile for the wild type and mutant IL-23heterodimers was measured by differential scanning calorimetry (DSC)using a MICROCAL® VP-capillary DSC instrument. 0.7 mg/ml protein samplesin PBS (7 mM Na₂HPO₄, 3 mM NaH₂PO₄, 130 mM NaCl, pH 7.1) were scannedfrom 10-100° C. at 90 o/hr, and the resulting thermograms were subjectedto a buffer blank subtraction and fitting procedure. The denaturation ofeach molecule was characterized by two unfolding transitions and thefitted transition midpoint (Tm) values of each transition were within1-4° C. of the wild type. The results show that none of the alanineshave mutants nor the 14 single alanine mutants show significant thermaldestabilization relative to the wild type.

Fourier Transform Infrared Spectroscopy (FT-IR) Analysis of IL-23Mutants

Secondary structure comparison of the alanine shave mutants and wildtype of IL-23 was performed using a FT-IR spectroscopy on Biotools ProtaFT-IR instrument with CaF₂ windows with a pathlength of ˜7 μm and aNe—He laser at 632.8 nm. Data were collected with a resolution of 2 cm⁻¹and analyzed with Prota/Bomem-GRAMS/31 AI software. Duplicatemeasurements were made for each sample and the method variability isabout 3%. Secondary structure content was calculated using Amide I peakas it is structure sensitive. Approximately an equal quantity of α-Helixand β-sheet was observed in all IL-23 samples as indicated by peaks at1637 cm-1 for α-Helix and peak at 1637 cm-1 as well as shoulder at 1687cm-1 for β-sheet. Overall no significant difference in FT-IR spectrumand calculated secondary structure result was observed in the alanineshave mutants compared to the wild type IL-23.

Circular Dichroism (CD) Analysis of IL-23 Mutants

Secondary structure comparison of the alanine shave mutants and wildtype of IL-23 using CD spectroscopy was performed using a Jasco J-815Spectrophotometer. The spectra were collected at 0.25 mg/mL IL-23protein concentration in PBS pH7.1 using a 1 mm path length cells at 25°C. from 300-190 nm with a data interval of 0.1 nm, a 50 nm/min scanningspeed, a 1 nm bandwidth, and 2 accumulations. Overall no significantdifference in secondary structure profile was observed in the alanineshave mutants compared to the wild type IL-23 using circular dichroism.

Nuclear Magnetic Resonance (NMR) Spectroscopy Analysis of IL-23 Mutants

Proton NMR is a highly sensitive technique that allows one to assess theconformational state at atomic detail. 1D ¹H NMR spectra were acquiredfor each of four mutant (M5, M7, M9, M10) and wild type IL23 proteins.All proteins were dialyzed simultaneously against NMR buffer (PBS in 8%D₂O/92% H₂O) to eliminate potential differences resulting from samplepreparation. In addition, ¹H signal intensities were corrected fordifferences in protein concentration by normalizing to the wild typespectrum. All NMR data was collected at 32° C. on a Bruker Avance 3spectrometer operating at 600 MHz. 1D NMR spectra were acquired usingthe standard bruker pulse sequence (ZG) optimized for solvent andexcipient suppression using the Watergate, WET, and Water flipbackselective excitation pulse schemes. Two thousand forty-eight (2048)scans were signal averaged for each spectrum. Cosine squared apodizationwas applied prior to Fourier transformation, and a first orderpolynomial baseline correction was used to flatten the appearance of thebaseline. Examination of each of the spectra for the individual proteinsreveals that each mutant protein was properly folded, as evidenced bythe well-dispersed resonances in both the high field (<0.5 ppm) and lowfield (>6.5 ppM) regions of the spectra. The high field methylresonances indicated the presence of an intact hydrophobic core; thedownfield amide protons reflected the existence of well-formed secondarystructure (alpha helices and beta sheets). Comparison of the spectrawith that for wild type IL23 indicated a very close match, precludingthe existence of large conformational changes in the protein structureinduced by the amino acid substitutions. In addition, the NMR resultsalso indicated that extra loss in activity in MS is unlikely due to anextra large disruption in structural integrity at the mutation sites inMS. The fact that MS was considerably closer to the wild-type protein byprinciple component analysis than M9 suggests, that the followingMS-mutations which are missing in M9, i.e., H53A, MS4A and E112A do notcause much of a disruption in the MS-structure. It was also observedthat mutants M7 & M9 which contain the elimination of an aromaticresidue appeared most similar to each other in the principle componentanalysis. All single mutants appear similar to wild-type with theexception of D118A and MS4A, however other controls demonstrate thatthese mutants maintain stability and activity similar to wild-type.

Summary of IL-23 Epitope Analysis

The methods of Alanine Shave and Single Mutagenesis have been used tomap the epitope of hIL-23 for the 7B7 Fab contained in both the 7B7 Fab,7B7 antibody and biAb3. The targeted mutagenesis strategy was performedusing epitope information generated by proteolytic digest LCMS analysis,peptide binding, hydrogen/deuterium exchange mass spectrometry, solventaccessible surface area calculations and docking algorithm calculations.Small scale screening by SPR and scale-up of select purified mutantsdemonstrated a specific epitope for 7B7/biAb3 binding hIL-23. The MSmutant shows dramatic decrease in binding to 7B7/biAb3, whilemaintaining functional activity, binding to p40 and p19 specificreagents, monomeric oligomeric state, thermal stability, and secondarystructural features similar to the wild-type. The M5 mutant showed adramatic loss of binding to 7B7/biAb3 while M9 & M10, which eachcontained two of the same mutated residues as M5, each show partial lossof binding to 7B7/biAb3. The affinity of the M5 IL-23 mutant was ≧300 nMfor 7B7/biAb3 which is approximately 1,500-fold weaker than wild-typeIL-23 demonstrating that the residues in the M5 mutant define theepitope for 7B7/biAb3 binding to IL-23. Thus, the data suggests that the7B7/biAb3 antibody binds to a discontinuous epitope on the p19 subunitof IL-23. This discontinuous epitope on IL-23p19 comprises at least twoepitope regions (region A (amino acid residues 33-59 of SEQ ID NO:6) andregion B (amino acid residues 89-125 of SEQ ID NO:6)). Specifically,with respect to the first epitope or region A of IL-23p19, amino acidresidues 54 (Met) and 55 (Asp) of SEQ ID NO:6 contribute a significantamount of binding energy to 7B7/biAb3's ability to bind IL-23p19. Withrespect to the second epitope or region B of IL-23p19, amino acidresidue 116 (Leu) is the primary residue within Region B energeticallycontributing to 7B7/biAb3's ability to bind IL-23p19.

Example 8 Marmoset EAE Model

Background and Rationale

Multiple sclerosis (MS) is a chronic autoimmune, inflammatory,neurodegenerative disease of the central nerve system (CNS)characterized by a loss of myelin in the brain and spinal cord. Althoughthe mechanisms underlying disease initiation are not clearly understood,the disease processes that contribute to clinical progression ofmultiple sclerosis are inflammation, demyelination, and axonal loss, orneurodegeneration. Macrophages and microglia are the main immune cellsof the CNS. These cells, as well as T cells, neutrophils, astrocytes,and microglia, can contribute to the immune-related pathology of, e.g.,multiple sclerosis. Furthermore, T cell reactivity/autoimmunity toseveral myelin proteins, including myelin basic protein (MBP),proteolipid protein (PLP), myelin oligodendrocyte protein (MOG), andperhaps other myelin proteins, have been implicated in the induction andperpetuation of disease state and pathology of multiple sclerosis. Thisinteraction of autoreactive T cells and myelin proteins can result inthe release of proinflammatory cytokines, including TNF-alpha,IFN-gamma, and IL-17, among others. Additional consequences are theproliferation of T cells, activation of B cells and macrophages,upregulation of chemokines and adhesion molecules, and the disruption ofthe blood-brain barrier. The ensuing pathology is a loss ofoligodendrocytes and axons, and the formation of a demyelinated“plaque”. The plaque consists of a lesion in which the myelin sheath isnow absent and the demyelinated axons are embedded within glial scartissue. Demyelination can also occur as the result of specificrecognition and opsinization of myelin antigens by autoantibodies,followed by complement- and/or activated macrophage-mediateddestruction. It is this axonal loss and neurodegeneration that isthought to be primarily responsible for the irreversible neurologicalimpairment that is observed in progressive multiple sclerosis.

Multiple sclerosis (MS) is classified into four types, characterized bythe disease's progression.

(1) Relapsing-Remitting MS (RRMS).

RRMS is characterized by relapse (attacks of symptom flare-ups) followedby remission (periods of recovery). Symptoms may vary from mild tosevere, and relapses and remissions may last for days or months. Morethan 80 percent of people who have MS begin with relapsing-remittingcycles.

(2) Secondary-Progressive MS (SPMS).

SPMS often develops in people who have relapsing-remitting MS. In SPMS,relapses and partial recoveries occur, but the disability doesn't fadeaway between cycles. Instead, it progressively worsens until a steadyprogression of disability replaces the cycles of attacks.

(3) Primary Progressive MS (PPMS).

PPMS progresses slowly and steadily from its onset. There are no periodsof remission and symptoms generally do not decrease in intensity. About15 percent of people who have MS have PPMS.

(4) Progressive-Relapsing MS (PRMS).

In this relatively rare type of MS, people experience both steadilyworsening symptoms and attacks during periods of remission.

Mayo Clinic (located on the internet atmayoclinic.org/multiple-sclerosis/types).

There is a large amount of clinical and pathological heterogeneity inthe course of human multiple sclerosis. Symptoms most often beginbetween the ages of 18 and 50 years old, but can begin at any age. Theclinical symptoms of multiple sclerosis can vary from mild visiondisturbances and headaches, to blindness, severe ataxia and paralysis.The majority of the patients (approximately 70-75%) haverelapsing-remitting multiple sclerosis, in which disease symptoms canrecur within a matter of hours to days, followed by a much slowerrecovery; the absence of symptoms during stages of remission is notuncommon. The incidence and frequency of relapses and remissions canvary greatly, but as time progresses, the recovery phases can beincomplete and slow to occur. This worsening of disease in these casesis classified as secondary-progressive multiple sclerosis, and occurs inapproximately 10-15% of multiple sclerosis patients. Another 10-15% ofpatients are diagnosed with primary-progressive multiple sclerosis, inwhich disease symptoms and physical impairment progress at a steady ratethroughout the disease process.

Both IL-23 and IL-17 are overexpressed in the central nervous system ofhumans with multiple sclerosis and in mice undergoing an animal model ofmultiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Theoverexpression is observed in mice when the EAE is induced by eithermyelin oligodendrocyte glycoprotein (MOG) 35-55 peptide- or proteolipidpeptide (PLP). Furthermore, neutralization of either IL-23/p19 or IL-17results in amelioration of EAE symptoms in mice (Park et al, NatImmunol. 6:1133 (2005); and Chen et al, J Clin Invest. 116:1317 (2006)).

Methods

Experimental autoimmune encephalomyelitis (EAE) is a well-characterizedand reproducible animal model which replicates certain aspects of MS.EAE is inducible in rodents and non-human primates, such as the commonmarmoset. Genain, C. P. and Hauser, S. L., “Creation of a model formultiple sclerosis in Callithrix jacchus marmosets,” J. Mol. Med.,75:187-197 (1997). In marmoset EAE, animals are immunized with arecombinant human myelin oligodendrocyte glycoprotein (rHuMOG) to inducethe development of a disease closely resembling human multiplesclerosis. Published studies of this model have employed MOG emulsifiedin complete Freund's adjuvant (CFA) as the immunogen, in a singleinoculation. Our early attempts to induce EAE in the marmoset using thepublished methodology led to onset of clinical symptoms between 23 to142 days after MOG injection (data not shown). Since this timeline wasconsidered to be too long for pre-clinical efficacy studies, a modifiedprotocol employing an initial priming dose followed by a boosterimmunization to induce disease was employed. Using the prime/boostprotocol as described in this study, we evaluated the activity of asurrogate bispecific antibody having an IL-17A/F binding entity and anIL-23 binding entity in this non-human primate model of EAE. Thesurrogate bispecific antibody was a biAbFabL (see FIG. 2), having thesame IL-17 A/F binding entity as biAb-1, biAb-2, biAb-3 and biAb-4,while having a surrogate IL-23 binding entity as the IL-23 bindingentity of biAb-3, for example, has reduced affinity for marmosetIL-23p19. Accordingly, an alternative IL-23 binding entity was utilizedin the surrogate bispecific antibody. The alternative IL-23 bindingentity utilized in the surrogate bispecific antibody was the same IL-23binding entity identified as IL-23 mAb in FIG. 13 and FIG. 14.

For induction of EAE, rHuMOG (BlueSky Biotech, Worcester, Mass.) wasdiluted in sterile PBS to 0.66 mg/ml and then emulsified with an equalvolume of incomplete Freund's adjuvant (IFA, Sigma #F5506) containing 5mg/mL of M. tuberculosis H37 RA (Difco #231141). 300 μL of the finalCFA/MOG mixture (containing 0.33 mg/mL of MOG and 2.5 mg/ml of H37RA)was injected at 2 sites on the shaved shoulder area of each animal (150μl×2 injection sites). All marmosets were immunized with CFA/MOG on thesame day (Day 0).

On Day 21, the animals received a booster immunization of IFA/MOG,(prepared as above but without the H37 RA) injected at 2 injection sitesover the shaved lumbar/hip area. Marmosets were anesthetized withKetamine (15 mg/kg) via IM injection for the prime and boostinoculations.

The surrogate IL-23/17 AF bAb (IgG4.1 k) was formulated in 20 mMsuccinic acid, 150 mM arginine buffer (pH 5.6). Dosing was started 1 dayprior to the priming inoculation with CFA/MOG. Animals were given eitherplacebo (PBS, SC, 2×/week) or IL-23/17 AF bAb (7 mg/kg, SC, 2×/week).Other treatment groups included BMS-938790 (IL-23 adnectin; 3 mg/kg, SC,2×/week), ADX_PRD1651 (IL-23/17 binectin, 10 mg/kg, SC, 2×/week), and asurrogate IL-23 mAb IgG4.1 (IL-23.15-g4P, 9 mg/kg, SC, 2×/week). Doseswere administered on Tuesdays and Fridays throughout the study period.Individual body weights were recorded weekly and animals were dosedbased on individual body weight. All SC doses were administered into theflank area following topical swabbing with alcohol. The dose sites werealternated (left or right side) for each dose administration. Treatmentgroups at the start of the study consisted of 8 animals per group (mixedmales and females). Animals were conscious and hand-restrained at thetime of drug injection.

Visual assessments of disease status were made daily beginning on Day12. Assessments were made and recorded by consensus of twoinvestigators. Clinical scores were based on a modification of publisheddisease score as described in Table 32 below.

TABLE 32 Marmoset EAE clinical scoring system 0 = No Clinical signs 0.5= reduced alertness/slow movements, or losing appetite 1.0 = weightloss >10% (initial wt. at beginning of study Day 0) 1.5 = unilateral orbilateral visual defects, dissociated gaze abnormalities 2.0 = visionimpairment and/or balance is weak, ataxia, or abnormal gait 2.5 = monoor paraparesis of front or back limbs and/or sensory loss and/or brainstem syndrome 3.0 = hemi- or paraplegia (paralysis of the posterior halfof one side) (can also include 1 leg and 1 hand paralysis at same time)4.0 = quadriplegia 5.0 = moribund or spontaneous deathResults

The number of animals with signs/symptoms of EAE disease per groups wasas follows:

Group A (PBS):

5 of 7 marmosets (71%) showed EAE-like disease and/or neurologicdysfunction at some point during the study. Disease presentationconsisted of blindness and paralysis.

Group B (IL-23 Adnectin, BMS-938790):

1 of 6 marmosets (17%) showed EAE-like disease. Disease presentationconsisted of progressive blindness and paralysis.

Group C (IL-23/17 Binectin, ADX_PRD1651):

6 of 7 marmosets (86%) showed EAE-like disease. However, 3 of theanimals showed evidence of EAE signs/symptoms on only a singleobservation day.

Group D (IL-23 mAb):

3 of 6 marmosets (50%) showed EAE-like disease. Blindness was prevalent.

Group E (IL-23/17 AF bAb):

4 of 8 marmosets (50%) showed EAE-like disease; 3 of these 4 animals hadtransient symptoms with a “positive” score on only a single observationday. Disease consisted of mostly mild symptoms such as slow movement orreduced alertness.

Compared to the disease incidence in Group A (PBS), the difference indisease incidence in each of the other treatment groups was notstatistically significant (p>0.05, Fisher's Exact Test).

A small subset of animals in the study did develop more severesigns/symptoms of EAE, with several marmosets requiring euthanasia tocomply with pre-established humane endpoints. The number of animals ineach group requiring euthanasia was as follows: Group A (2); Group B(1); Group C (1); Group D (0); Group E (0).

Animals that were euthanized for humane reasons as a result of EAEsymptoms were assigned a clinical severity score of “4” at the time ofeuthanasia which was carried throughout the remainder of the study forthe purpose of calculating a group mean clinical severity score. FIG. 12depicts the mean clinical severity score for each treatment group overthe course of the study. Group A (PBS) reached a peak score ofapproximately 1.6 by the end of the study. All other groups displayedlower mean clinical scores, with Group E (IL-23/17 AF bAb) showing onlya brief period (˜1 wk) when any signs of EAE disease were evident. Dueto the limited numbers of animals in each group and the inter-animalvariability in EAE disease scores among animals within a group, none ofthe differences between Group A (PBS) and the other treatment groups wasstatistically significant (Mann-Whitney U Test).

In summary, this study showed a trend toward reduced disease severityand reduced disease incidence in the marmoset EAE model by several ofthe compounds evaluated. Overall, the animals treated with IL-23/17 AFbAb (IgG4.1 k) appeared to experience the most beneficial outcome (FIG.12), though the differences between groups were not statisticallysignificant. The finding that the animals treated with the IL-23/17 AFbAb were better protected than the animals treated with just the IL-23mAb, suggests that dual targeting of both the IL-23 and IL-17AF pathwayswill result in greater efficacy in the treatment of human diseases inwhich these cytokine pathways play a role, including but not limited tomultiple sclerosis.

Post-Mortem MRI

At the end of the study, the surviving marmosets were submitted fornecropsy. The skull cap was removed and the brain was fixed in situ informalin for 3 weeks. To assess lesions in white matter and the optictracks, T2W and proton density MRI scans were conducted with the BrukerBiospec 7T system with a 72 mm Quad RF coil, using the followingparameters: Total scan time ˜15-20 mins per sample, 23 axial images werecollected, TR/TE=5000/20 ms, slice thickness=1.2 mm, FOV=4 cm,matrix=256×256, in-plane resolution=156 mm².

Scans were reviewed and semi-quantitative interpretations were performedby consensus reading of 3 radiologists after review of all MRI imagesfor each group of animals. Lesion scoring was based on lesion count inthe white matter covering the entire brain. Optic nerve scoring wasbased on swelling and increased signal intensity reflecting inflammationin the optic tract and nerve.

Overall, a significant (p<0.05) reduction in lesion load was observedfor the IL-23/17 AF bAb group compared to the vehicle group (FIG. 13).The optic tract score was also significantly lower in the IL-23/17 AFbAb group compared to vehicle (FIG. 14). None of the other treatmentgroups were significantly different than vehicle-treated group foreither of these MRI measurements. Thus, consistent with the clinicaldisease scores, the group of animals treated with the IL-23/17 AF bAbhad the lowest mean MRI values again suggesting that dual targeting ofboth the IL-23 and IL-17AF pathways will result in greater efficacy inthe treatment of diseases in humans.

Example 9 IL-17A and IL-17F Epitope Mapping

The analysis described in this Example 9 aims to identify the epitopicresidues on IL-17A and IL-17F for which the IL-17A/F binding entity ofbiAb3 (heavy chain variable domain as shown in SEQ ID 13 and light chainvariable domains as shown in SEQ ID NO:9) binds.

The strategy to identify the key differences between the epitope of thebiAb3 and the mouse parent (339.15.5.3) utilized X-ray crystallography,site-directed mutagenesis, in-silico mutagenesis, and binding andfunctional assays to analyze the mutants. The heavy chain variabledomain of the IL-17A/F binding entity of biAb3 is a humanized version ofthe heavy chain variable domain of clone 339.15.5.3, clone 339.15.3.6 orclone 339.15.5.3. The heavy chain CDR residues of the IL-17A/F bindingentity of biAb3 and clone 339.15.5.3, clone 339.15.3.6 or clone339.15.5.3 are identical. However, the two mAbs differ in the frameworkregion of their heavy chain variable domains. The two mAbs havedifferent light chains therefore the residues of IL-17 in contact withthe light chain of each mAb was the focus of this study.

X-Ray Crystallography

IL17A Fab complex and IL17F Fab complex crystallography was performed todetermine the contact interface residues for contact surface modelingfor epitope/paratope residue predictions, and buried surfacedetermination. The three-dimensional structures were also used forProtein Mutant energetic calculations based on modeled crystal structurecomplexes.

The Fabs of each antibody were cloned and expressed periplasmically inE. coli strain BL21 (humanized 339-134) or BL21 Star (BiAb).Purification for both included IMAC followed by SEC. Both IL-17A andIL-17F were expressed in E. coli strain W3110 as inclusion bodies andrefolded, followed by a several step purification.

The Fab derived from the IL-17 arms of the biAb3 (heavy chain variabledomain of SEQ ID NO:13 and heavy chain CH1 domain of SEQ ID NO:15; and alight chain of SEQ ID NO:17) and the Fab derived from the humanized leadof the parent (humanized anti-human IL-17A/F antibody 339-134 mAb (SEQID NO:65 and SEQ ID NO:67) were complexed with either human IL-17A orIL-17F. Complex formation of either IL-17A or IL-17F with the BiAb3 andthe humanized anti-human IL-17A/F antibody 339-134 mAb was monitored bySEC.

Co-crystals of each of the four complexes were generated by broadscreening followed by optimization of crystallization conditions. A dataset on each sample was collected at the APS LS-CAT beamlines.

TABLE 33 Summary of X-ray crystallography dataset resolution. ComplexResolution IL-17A A2999F/Parent Fab A3052F 2.85 Å IL-17F A2768F/ParentFab A3052F 3.75 Å IL-17A A2999F/Lead Fab A3185F  3.4 Å IL-17FA2768F/Lead Fab A3185F 4.25 Å

Each structure was determined by molecular replacement using Phaser MRfrom the CCP4 suite. The IL-17A search model was derived from PDB ID2VXS. The IL-17F search model was taken from PDB ID 1JPY. For each Fab,search models were generated for the constant and variable domains usingthe humanized Fab from PDB ID 3IDX with the hypervariable loops deleted.The final models were obtained after iterative rounds of refinement inREFMAC5 and manual model building in Coot. In addition, additionalrounds of refinement and map generation in Autobuster were performed formodel building and refinement of all of the lower resolution structures.Crystallographic statistics for data collection and refinement are shownfor the humanized parental Fab structures in Table 34 and the humanizedlead Fab structures in Table 35. Each structure was assessed forgeometry using MolProbity which had been downloaded to run on aninternal server.

TABLE 34 Crystallographic statistics for the humanized parental FabA3052F in complex with either IL-17A or IL-17F IL-17A/Parent Fab A3052FIL-17F/Parent Fab A3052F Overall Highest shell Overall Highest shellCrystal ID 238318e7 238699f10 Unique puck ID oyg0-1 apa7-6 Collectiondate 1 Nov. 2012 18 Dec. 2012 Δφ 0.5° 1.0° Images 71-260 (95°) 1-180(180°) Wavelength 0.97856 Å 0.97856 Å Space Group P3₂21 C2 Unit Cell a =b = 141.9 Å, c = 91.2 Å a = 226.1 Å, b = 62.3 Å, c = 117.3 Å α = β =90°, γ = 120° α = γ = 90°, β = 104.4° Solvent content  70% 60%   V_(m)4.1 Å³/Da 3.1 Å³/Da Resolution 50-2.85 Å 2.91-2.85 50-3.75 Å 3.84-3.75I/σ 18.2 2.3 16.7 2.0 Completeness 99.1%  99.3% 97.4% 97.2% R_(merge)0.062 0.538 0.066 0.709 Multiplicity 5.9 6.0 3.4 3.5 Reflections 24,8281821 16,223 1198 Mosaicity 0.4 0.3-0.8 Refinement R 0.269 0.251 R_(free)0.309 0.300 Validation Ramachandran favored 91.9%  86.8% Ramachandranoutliers 1.0%  3.6% Rotamer outliers 3.2% 10.3% Clash score 3.78(100^(th)) 10.08 (97^(th)) Molprobity score 2.04 (99^(th)) 2.92(91^(st))

TABLE 35 Crystallographic statistics for the humanized lead Fab A3185Falone or in complex with IL-17A or IL-17F Lead Fab IL-17A/Lead FabIL-17F/Lead Fab A3185F A3185F A3185F Highest Highest Highest Overallshell Overall shell Overall shell Crystal ID 243072a6 240719f6 238860g7Unique puck ID koc5-3 jsm6-5 cum1-2 Beamline APS 21 ID-D APS 21 ID-D APS21 ID-G Collection date 18-April-2013 18-April-2013 30-Nov-2012 Δφ 1.0°1.0° 0.5° Images 1-257 (257°) 1-180 (180°) 1-180 (180°) Wavelength0.93005 Å 0.93005 Å 0.97856 Å Space Group C2 C222₁ P2₁ Unit Cell a =92.1 Å, b = a = 54.6 Å, b = 83.6 Å, a = 115.0 Å, b = 61.8 Å, 60.1 Å, c =73.0 Å, c = 248.9 Å, α = β = c = 124.9 Å, α = γ = α = γ = 90°, γ = 90°90°, β = 92.3° β = 94.9° Solvent content   39%   51%   64% V_(m) 2.0Å³/Da 2.5 Å³/Da 3.4 Å³/Da Resolution 50-2.1 Å 2.14-2.10 Å 50-3.4 Å3.48-3.40 Å 50-4.25 Å 4.35-4.25 Å I/σ 13.2 3.9 18.6 3.1 13.7 2.8Completeness 99.6% 99.4% 98.1% 97.5% 98.2% 98.9% R_(merge) 0.108 0.4500.090 0.583 0.066 0.556 Multiplicity 5.2 5.3 4.5 4.9 3.6 3.7 Reflections23,262 1708 9200 653 12,522 931 Mosaicity 0.3 0.5 0.3 Refinement R 0.1750.243 0.285 R_(free) 0.227 0.313 0.347 Validation Ramachandran 97.3%94.1% 92.5% favored Ramachandran  0.0%  0.8%  1.4% outliers Rotameroutliers  0.8%  5.3%  4.4% Clash score 2.91 (99^(th)) 3.40 (100^(th))3.98 (100^(th)) Molprobity score 1.22 (100^(th)) 2.08 (100^(th)) 2.14(100^(th))

Each Fab primarily bound to one half-site of the IL-17 homodimerprimarily through its heavy chain, and to a lesser extent through thelight chain. Differences in binding of the humanized parent andhumanized lead Fab appear consistent with higher affinity of thehumanized lead Fab.

Globally, each of the IL-17/BMS Fab complexes exhibited the same overallstructure in which one Fab recognized one half-site of the IL-17homodimer (FIG. 15). Thus, the binding stoichiometry is showncrystallographically to be two (2) Fabs to one (1) IL-17 homodimer (ortwo (2) Fabs to two (2) IL-17 protomers). The majority of theinteractions with the interleukin are formed by the heavy chain with thehypervariable loop3 (or complementary determining region CDR3) formingthe heart of the antibody-antigen interaction. In addition, a number ofresidues of CRD2 of the heavy chain interact with the interleukin. CDR1of the heavy chain does not appear to interact with the interleukinsignificantly. For the light chain, CDR3 is involved in the recognitionof IL-17. CDR1 of the light chain also appears to provide a weak, longerrange binding interaction.

The crystal structure coordinates were used to define the contactinterface as previously described (S. Sheriff, “Some Methods forExamining the Interaction between Two Molecules,” Immunomethods,3:191-196 (1993)), where a minimal definition, defined as residues incontact, was derived from the program CONTACSYM (Sheriff, S.,Hendrickson, W. A., and Smith, J. L. (1987). Structure of Myohemerythrinin the Azidomet State at 1.7/1.3 Å Resolution. J. Mol. Biol. 197,273-296.). A maximal definition of the interface, defined as residues atleast partially buried by the interaction, was derived from Program MS(Connolly, M. L. (1983). Analytical Molecular Surface Calculation. J.Appl. Crystallogr. 16, 548-558).

TABLE 36 Contact and Buried Interface Residues of Complexes IL17 TypeIL17A (SEQ ID NO: 2) IL17F (SEQ ID NO: 4) Humanized Humanized HumanizedHumanized mAb Type Parent Lead Parent Lead Resolution 2.85 Å 3.4 Å 3.75Å 4.2 Å Chain A B A B Asn 75 Asn 83 Asn 83 Asn 83 Ala 92 Ala 92 Ala 100Ala 100 Ala 100 Ala 100 *Lys 93 *Lys 93 *Gln 101 *Gln 101 *Gln 101 *Gln101 *Cys 94 *Cys 94 *Cys 102 *Cys 102 *Cys 102 *Cys 102 *Arg 95 *Arg 95*Arg 103 *Arg 103 *Arg 103 *Arg 103 *His 96 *His 96 *Asn 104 *Asn 104*Asn 104 *Asn 104 *Leu 97 *Leu 97 *Leu 105 *Leu 105 *Leu 105 *Leu 105*Gly 98 Gly 106 Asp 103 Lys 113 Lys 113 *Val 106 Val 106 *Glu 114 *Glu114 *Glu 114 *Glu 114 Asp 107 Asp 107 Asp 115 Asp 115 Asp 115 Asp 115*Tyr 108 *Tyr 108 *Ile 116 *Ile 116 *Ile 116 *Ile 116 *His 109 *His 109Ser 117 *Ser 117 *Ser 117 *Ser 117 Met 118 Met 118 *Asn 111 *Asn 111*Asn 119 *Asn 119 *Asn 119 *Asn 119 *Ser 112 *Ser 112 *Ser 120 *Ser 120*Ser 120 *Ser 120 *Val 113 *Val 113 *Val 121 *Val 121 *Val 121 *Val 121*Pro 114 *Pro 114 Pro 122 Pro 122 *Pro 122 *Pro 122 *Gln 124 Gln 124*Gln 124 Ser 141 Ser 141 Thr 149 Thr 149 Thr 149 Thr 149 Val 147 *Val147 Val 155 Val 155 Val 155 Thr 148 Thr 156 Thr 156 Thr 156 Thr 156 *Pro149 *Pro 149 *Pro 157 *Pro 157 *Pro 157 *Pro 157 *Ile 150 *Ile 150 *Val158 *Val 158 Val 158 *Val 151 *Val 151 His 152 *His 152 His 153 His 153Residues with an asterick (*) are in contact at the complex interface.Residues lacking an asterick are completely buried in the complexcontact interface.

The main interaction of the Fab with IL-17A (SEQ ID NO:2) appears to beto residues L97 and a stretch of residues from H109-N111 (FIG. 15).I100, G98, N111, S112, V113, and P114 are conserved between IL-17A andIL-17F (amino acid residues I100, G98, N111, S112, V113, and P114 of SEQID NO:2 or IL-17A correspond to amino acid residues I108, G106, N119,S120, V121, and P122 of SEQ ID NO:4 or IL-17F). Thus, after obtainingthis initial structure, it was expected that the humanized parent FabA3052F would bind IL-17F in essentially an identical manner as thatobserved with IL-17A. However, near these residues there are severalresidues which differ between IL-17A (SEQ ID NO:2) and IL-17F (SEQ IDNO:4), such as K93 in IL-17A (Q101 in 17F), H95 in IL-17A (N104 in 17F),Y108 in IL-17A (I116 in 17F), and H109 in IL-17A (S117 in 17F). TheC-terminus of IL-17A (SEQ ID NO:2) also appears to weakly interact withthe Fab through residues P149 and I150, which correspond to residuesP157 and V158 of IL-17F (SEQ ID NO:4). However, the differences betweenIL-17A and IL-17F would not be expected to alter the globalantibody-antigen interaction significantly.

In comparison with the 2.85 Å resolution of the humanized parent Fab,the 3.4 Å resolution of the humanized lead Fab exhibits very similarinteractions between the heavy chain and the interleukin as expectedgiven the same sequence for the heavy chain. In contrast, the lightchains between these two Fabs are completely different and as expected,these residues coordinate the interleukin in a different manner. ThebiAb Fab has one fewer residue in CDR3 which allows the loop to stretchout over the interleukin, allowing the backbone oxygen of G93 (G93 ofSEQ ID NO:9 or G4 of SEQ ID NO:27) of the biAb Fab to form a directhydrogen bond with the backbone nitrogen of Y108 of SEQ ID NO:2(IL-17A). The same atom (backbone oxygen of N91 of SEQ ID NO:67 in thehumanized parent Fab) was 2.3 Å away from the location in the parent Faband was unable to hydrogen bond with the interleukin. Furthermore, thechange of WN to YG allows Y33 of CDR1 (Y33 of SEQ ID NO:9) to approachthe interleukin by 5.1 Å relative to its position in the lower affinityparent Fab thereby gaining additional packing interactions with H109 ofIL-17A (SEQ ID NO:2). Finally, Y96 (Y96 of SEQ ID NO:9) of the biAb Fabmay form a hydrogen bond with N106 (N106 of SEQ ID NO:13) of the heavychain, assisting to align it in a hydrogen bond with the Y108 (Y108 ofSEQ ID NO:2) backbone oxygen of the interleukin (IL-17A). Overall, thesechanges in the interface of CDR3 of the biAb Fab appear consistent withits higher affinity and have been tested by site-directed mutagenesis asshown below. Unfortunately, crystal structures were not obtained for acomplex consisting of the mouse parental Fab with either IL-17A orIL-17F. This suggests that the conditions for optimizing theinteractions of these complexes are different from those used forsuccessful crystallization of the humanized parental and lead Fabs.

In addition to interpretation of individual residue contacts, anothermeasure of the differences in the humanized parent Fab vs. the biAbbound to the interleukin can be captured by calculating the totalsurface area buried by the interacting regions of the IL17/Fabcomplexes. This analysis was carried out using the MS algorithm for theIL-17A Fab complexes, as they were the highest resolution, and shouldprovide the most reliable comparison. The two structures with IL-17Fwere over 3.5 Å and not considered reliable for this analysis. Theparental Fab buried 720 Å² on IL-17A, while the biAb Fab buried 820 Å²(see Table 37). This difference (100 Å²) in surface area is supported bythe measured increased binding affinity of the lead Ab for IL-17A.Interestingly, the surface area of due to an extended side chainconformation for many amino acids is less than 100 Å²(A,N,D,C,G,L,P,S,T,V), see Atlas of Protein Side-Chain Interactions V1.Singh and Thornton 1992, 6-11). So, in aggregate by this measurement,the binding epitope difference on IL-17A for the lead vs. the parentalstructures might be described as approximately one (1) residueequivalent.

TABLE 37 Surface Area Complex IL-17A/Parent IL-17A/Lead Resolution 2.85Å 3.4 Å Buried on IL-17A 720 820 Buried on FAB 740 830Computational Epitope Prediction and Design of Alanine Shave Mutants

To provide detailed characterization of the different contributions ofIL-17A and IL-17F epitope residues to the binding kinetics of theParental and Lead Fabs, residues in the binding interfaces identified byX-ray crystallography were selected for site specific mutation tofurther distinguish differences between the binding of the human parentmAb and biAb.

A variety of criteria for selection of individual and multiple mutationson a single molecule were employed to select a set of representative andinformative mutants to test. Crystal structures of IL-17A/Parental Fab,IL-17F/Lead Fab, described earlier herein were used to inform selectionof residues to mutate. Residues at the ligand-Fab interaction interfacewere selected, while focusing on residues in contact with Fab lightchain residues. Regions of poor definition in the crystal structure forone Fab but not the other were also selected because t his may suggest adifference in residue mobility within the complex in parent vs. biAb. Inaddition, residue positions where homologous IL-17 A/F residues differwere also selected because this may indicate tolerance to change for Fabbinding, or may contribute to different binding affinities relative tothe parental and biAb Fabs. The interface was modeled with the proposedmutants to look for differences in energetics in complex with parentalvs. biAb Fabs. Clusters of contact residues (alanine shave mutants) werecombined to generate mutants with additive or synergistic changes inbinding affinity. The mutants of each interleukin which were generatedfor experimental analysis are shown in Table 38 and 39.

TABLE 38 IL-17A mutants IL17A-WT Residues 24-155 of SEQ ID NO: 2IL17A-M1 N105A, Y108A, H109A IL17A-M2 L97A, Y108A, N111A IL17A-M3 K61AIL17A-M4 K61A, S64A, R69A IL17A-M5 N105A IL17A-M6 Y108A IL17A-M7 H109AIL17A-M8 V106A, D107A IL17A-M9 I150A, V151A IL17A-M10 Y108A, H109A,V151A, H152A IL17A-M11 Y108A, V151A IL17A-M12 H109A, I150A, H152A

TABLE 39 IL-17F mutants IL17F-WT Residues 31-163 of SEQ ID NO: 4IL17F-M1 L105A, I116A, N119A IL17F-M2 K113A, E114A, I116A IL17F-M3 S69A,R72A, R77A IL17F-M4 S69A IL17F-M5 R72A IL17F-M6 K113A IL17F-M7 E114AIL17F-M8 I116A IL17F-M9 D115A, S117A IL17F-M10 V158A, I159ACloning & Expression of IL-17A & F Epitope Mapping Alanine Mutants

The mutant constructs of IL-17A and IL-17F were generated by genesynthesis and then cloned into the transient transfection vector forexpression in HEK293-6E cells. 30 mL cultures of HEK293-6E cells at1×10⁶ cells/ml were transfected with expression plasmids/PEI complex andcells harvested on 120 hours post transfection.

Biacore Concentration Analysis of IL-17A&F Mutants

The concentration of each alanine mutant of IL-17A and IL-17F in the HEKharvest supernatants was quantitated by the level of capture on ananti-his Fab Biacore sensor surface. Protein A and huIgG surfaces werealso immobilized as controls to assess non-specific binding ofsupernatants (no non-specific binding was observed). The concentrationin each supernatant was quantitated using a standard curve of purifiedIL-17A-his or IL-17F-his ranging from 80 ug/mL to 0.039 ug/mL. Thecalibration curve was fit to a linear curve for the data from 0.3125 to0.0039 ug/mL. The supernatants were run at 1:20, 1:60 and 1:180 dilutionto allow multiple measurements in the linear range of the standardcurve.

IL-17A mutant 8 had significantly reduced expression (only about 10% ofthe expression level of the wild-type). IL-17F mutants M2, M3, M7, andM9 had significantly reduced expression. The M9 mutant of IL-17F wasvirtually undetectable while the IL-17F M2 and M7 mutants were at lessthan 5% of the wild-type expression level.

TABLE 40 Expression levels of IL-17A and IL- 17F constructs in HEKsupernatants μg/mL expression level in IL-17 variant HEK supernatantsIL17A-WT 7.2 IL17A-M1 6.3 IL17A-M2 13.0 IL17A-M3 15.3 IL17A-M4 18.6IL17A-M5 10.3 IL17A-M6 11.7 IL17A-M7 12.4 IL17A-M8 0.6 IL17A-M9 13.6IL17A-M10 14.8 IL17A-M11 13.2 IL17A-M12 12.1 IL17F-WT 13.6 IL17F-M1 16.2IL17F-M2 0.3 IL17F-M3 1.4 IL17F-M4 12.2 IL17F-M5 6.8 IL17F-M6 7.1IL17F-M7 0.3 IL17F-M8 10.3 IL17F-M9 <0.1 IL17F-M10 15.0IL-17 Bioassay (NIH/3T3/KZ170 NE-κB Luciferase Reporter assay)

A murine fibroblast cell line (NIH/3T3, ATCC# CRL-1658) was stablytransfected with an NF-κB luciferase reporter designated KZ170 andcloned out. NIH/3T3/KZ170 clone 1 cells were seeded at 10,000 cells/wellin 96-well white opaque luciferase plates and incubated overnight at 37°C. The following day serial dilutions of recombinant human IL-17A,IL-17F, IL-17A Alanine mutants, and IL-17F Alanine mutants, using theBiacore determined concentrations, were made up in assay media and addedto the plates containing the cells and incubated at 37° C. for 4 hours.Following incubation the media was removed and cells lysed before beingread on the Berthold Centro XS³ Luminometer using flash substrateaccording to manufactures instructions. Increases in mean fluorescenceintensity (via activation of the NF-κB luciferase reporter) wereindicative of an IL-17A or IL-17F receptor-ligand interaction. EC₅₀(effective concentration at 50 percent) values were calculated usingGraphPad Prism®4 software for each IL-17A and IL-17F Alanine mutant.

All of the IL-17A mutants show cell functional activity, though somehave lost a few fold in activity with respect to wild-type (FIG. 16).All IL-17F mutants except M3 show cell functional activity, though 3 ofthe mutants (M2, M5, M10) show significantly reduced activity (FIG. 17).

Biacore Binding Analysis of IL-17A&F Mutants for Binding to the BiAb3and Other Related Antibodies

The antibodies used for the Biacore binding assay were the biAb3 (heavychain variable domain as shown in SEQ ID NO:7 and light chain variabledomain as shown in SEQ ID NO:9) and the mouse parent antibody 339.15.5.3(which contains the same variable region sequences as 339.15.3.5 and339.15.3.6). Isotyping using the IsoStrip Mouse Monoclonal AntibodyIsotyping Kit (Roche, Indianapolis, Ind., USA) demonstrates that the339.15.5.3 antibody is the IgG2a/kappa just like the 339.15.3.5 used forhumanization. No sequence or isotype differences have been determinedbetween 339.15.3.5 and 339.15.5.3.

The binding of the supernatants from the 30 mL expression of all 12IL-17A and 10 IL-17F alanine mutants and a wild-type control supernatantfor each (see Table 41 and Table 42) was measured by surface Plasmonresonance (SPR, Biacore)) on a Biacore T100 in HBS-EP (10 mM HEPES, 3 mMEDTA, 150 mM NaCl, 0.05% Tween 20, pH 7.4) at 25° C. The relevantantibodies and receptors were captured at a level of about 150-250 RUsby protein A immobilized at 3000 RUs on a CM5 sensor chip. In additionto the biAb3 and the parent mAb (339.15.3.5), other anti-IL-17 mAbs wereused as controls for domain binding. In addition, the commercialreceptor for IL-17A was used as a control: hIL-17RA-Fc (from R&DSystems) though a suitable IL-17RC reagent was not identified for thisassay. The supernatants were diluted based on the anti-his quantitationdetermined concentration into HBS-EP at a concentration of 9 nM anddiluted serially 1:3 and injected at 30 uL/min over the mAb or receptorsurface for 90 seconds and, after a dissociation time, regenerated with10 mM Glycine, pH 1.5. IL-17A M2 and IL-17F M1 were also run at higherconcentration (in the 400-500 nM range at the expression level of thesupernatant) because little to no binding signal was observed for thebiAb captured surface. Binding to a reference surface of Protein Awithout any captured antibody was subtracted from all specific bindingcurves before analysis. All titration curves were fit to a 1:1 Langmuirbinding model to determine the Kd values shown in FIG. 18 and Tables 41and 42.

The Biacore results demonstrate that the IL-17A mutants M1, M2, M6, M10,and M11 show a reduction in binding affinity for the BiAb and containresidues that contribute to the binding epitope differences comparedwith the parent antibody. Most of these mutants show a 3-15 foldreduction (45-fold for M1) in binding to the IL-17RA-Fc likely becausethe receptor binding site has been altered. However, the cellularpotency was maintained within a few fold for all mutants that impactedthe BiAb3 interaction suggesting that these receptor disruptions are notsignificant for function.

The IL-17F mutants M1, M2, M7, and M8 show a reduction in bindingaffinity for the biAb however these same mutants show a similar reducedbinding affinity for the parent antibody indicating that the epitopechange between parent and biAb in IL-17A does not translate to IL-17F.These four mutants maintain potency in the cell functional assay similarto wild-type IL-17F, however many of the other mutants do not whichlimits our interpretation of the IL-17F mutagenesis.

TABLE 41 Biacore Kinetic Analysis of IL-17A Alanine Mutants BindingbiAb3 and mouse parent antibodies biAb3 mouse parent Kd-shift ΔΔGKd-shift ΔΔG Variant Kd (nM) (from WT) (kcal/mole) Kd (nM) (from WT)(kcal/mole) WT 0.05 none 0 0.03 none 0 M1 0.23 4.5 0.9 0.02 none −0.3M2 >1 uM >20,000 >5.9 >1 uM >35,000 >6.2 M3 0.05 none 0 0.02 none −0.4M4 0.09 2 0.4 0.02 none −0.3 M5 0.04 none −0.2 0.01 none −0.9 M6 0.112.2 0.5 0.02 none −0.3 M7 0.04 none −0.2 0.01 none −0.4 M8 0.05 none 00.03 none 0.1 M9 0.04 none −0.1 0.01 none −0.4 M10 0.3 5.7 1.0 0.02 none−0.3 M11 0.11 2.2 0.5 0.01 none −0.4 M12 0.06 none 0.1 0.02 none −0.3

TABLE 42 Biacore Kinetic Analysis of IL-17F Alanine Mutants BindingbiAb3 and mouse parent antibodies biAb3 Mouse parent Kd-shift ΔΔGKd-shift ΔΔG Variant Kd (nM) (from WT) (kcal/mole) Kd (nM) (from WT)(kcal/mole) WT 0.08 none 0 0.005 none 0 M1 >1 uM >12,000 >5.6 >1uM >100,000 >7.3 M2 0.7 9.0 1.3 0.01 2 0.5 M3 0.15 2.0 0.4 <0.001 none−1.3 M4 0.08 none 0 <0.001 none −0.8 M5 0.11 none 0.2 <0.001 none −2.1M6 0.08 none 0 <0.001 none −1.6 M7 0.55 7.0 1.2 0.03 5.9 1.1 M8 0.2 2.50.6 0.02 3.7 0.8 M9 NM NM NM NM NM NM M10 0.09 none 0 <0.001 none −1.6NM—not measured because the mutant was not expressed at detectablelevels.In Silico Mutagenesis

Energetic analyses were preformed for the IL-17A-Parent Fab, IL-17A-LeadFab, and IL-17F-Lead Fab. Because the X-ray structures of the complexeswere incomplete protein modeling was used to complete the structuralmodels by building in the missing amino acid side chains using standardprotocols (Maestro protein preparation wizard and Prime side chainmodeling). The structural models were then used to calculate interactionenergies for wildtype protein (IL-17A or IL-17F) or mutant IL-17molecules with the Fabs. The interaction energies are a measure of thecalculated affinity of the Fab towards the IL-17 (treated as ligand). Inorder to calculate the stability and delta-stability of mutant proteinsthe software MOE (ver. 2012.10, Chemical Computing Group) was used. TheResidue Scanning protocol was used to perform computationalsite-directed mutagenesis to generate mutations and perform the affinityand stability calculations.

A comparison of the computational predicted binding energies with thecalculated ΔΔG values determined from the Biacore affinities for IL-17Amutants are shown in FIG. 19. The IL-17A mutants identified in FIG. 19are mislabeled. The SEQ ID NO:2 numbering of IL-17A mutants identifiedin FIG. 19 are one less than they should be. The mutants are asidentified as in Table 38. For example, N104A, Y107A and H108A should beN105A, Y108A and H109A and so on across the Figure. This comparisonshows that the trend across the panel of mutants for the Biacoreenergetics are in agreement with the computational energetic predictionof the binding interface. Analysis of the IL-17A binding to the mouseparent were not possible because that complex failed to crystallize andan acceptable model could not be generated. An attempt to generateresults for IL-17F mutants binding to the BiAb3 produced inconsistentresults, likely because the structures used for these were at poorresolution and missing necessary residue information.

Summary of IL-17A&F Epitope Mapping

The strategy to identify the key differences between the epitope of theBiAb3 and the mouse parent (339.15.5.3) utilized X-ray crystallography,site-directed mutagenesis, in-silico mutagenesis, and binding andfunctional assays to analyze the mutants. The two mAbs differ in theirlight chain therefore the residues in contact with the light chain ofeach mAb were the focus of this study.

The IL-17A mutagenesis resulted in a panel of mutants with goodexpression and reasonable cellular potency. A significant change in theΔΔG for binding to the biAb3 was measured for all mutants that containY108A with a 0.5 kcal/mole change measured for the Y108A single pointmutant. These changes in ΔΔG were not observed for binding to the mouseparent antibody indicating that the Y108 is new residue in the epitopeof IL-17A for the biAb3 compared with the mouse parent mAb. Theenergetic data is consistent with the interface analysis of the crystalstructures showing that this residue is interacting with the light chainwhich is different in biAb3 compared with the mouse parent and thatY108A is brought into closer proximity to the biAb3 due to differencesin the CDR3 of the biAb3 light chain compared with the mouse parent. Theonly IL-17A mutant shown here that impacts the binding of the mouseparent antibody is one that interacts with a heavy chain residueindicating that the light chain does not play much of a role in bindingof the mouse parent antibody to IL-17A.

The IL-17F mutagensis resulted in a panel of mutants with more variableand lower expression levels and significant losses in potency. Thecrystal structures obtained for IL-17F were also at poorer resolutionthan the IL-17A structures. This suggests that IL-17F is not as amenableto mutagenesis and potentially more dynamic in nature. However, themutants with reduced binding to the biAb3 also reduced binding to themouse parent mAb. And, all of the these mutants with reduced mAb bindingwere of reasonable potency and lower, but sufficient, expression levelsfor the analysis.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

What is claimed is:
 1. An isolated monoclonal antibody that specifically binds to IL-17A (SEQ ID NO:2) and IL-17F (SEQ ID NO:4) comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises a CDR1 having the amino acid sequence of SEQ ID NO:25, a CDR2 having the amino acid sequence of SEQ ID NO:26 and a CDR3 having the amino acid sequence of SEQ ID NO:27, and wherein the light chain variable domain comprises a CDR1 having the amino acid sequence of SEQ ID NO:22, a CDR2 having the amino acid sequence of SEQ ID NO:23 and a CDR3 having the amino acid sequence of SEQ ID NO:24.
 2. The isolated monoclonal antibody of claim 1, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:13.
 3. The isolated monoclonal antibody of claim 1, wherein the light chain variable domain comprises the amino acid sequence of SEQ ID NO:9.
 4. The isolated monoclonal antibody of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NOs:16, 18, 28, 29 or
 74. 5. The isolated monoclonal antibody of claim 1, wherein the light chain comprises the amino acid sequence of SEQ ID NO:17.
 6. The isolated monoclonal antibody of claim 1, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:74, and the light chain comprises the amino acid sequence of SEQ ID NO:17.
 7. The isolated monoclonal antibody of claim 1, wherein the antibody comprises a human constant region.
 8. The isolated monoclonal antibody of claim 7, wherein the isotype of the heavy chain is IgG1, IgG2, IgG3 or IgG4.
 9. The isolated monoclonal antibody of claim 8, wherein the IgG4 heavy chain has a Serine to Proline mutation at position 241 according to Kabat.
 10. A composition comprising the monoclonal antibody according claim 1 and a pharmaceutically acceptable carrier.
 11. A method of treating a disease characterized by elevated expression of IL-17A and/or IL-17F in a mammal in need of such treatment comprising administering a therapeutically effective amount of the monoclonal antibody according to claim 1 or the composition of claim 10 to said mammal.
 12. The method of claim 11, wherein the disease is irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, atopic dermatitis, contact dermatitis, systemic sclerosis, systemic lupus erythematosus (SLE), antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV), giant cell arteritis, multiple sclerosis (MS), relapsing-remitting multiple sclerosis, secondary-progressive multiple sclerosis, primary-progressive multiple sclerosis, progressive-relapsing multiple sclerosis, colitis, arthritis, rheumatoid arthritis (RA), osteoarthritis, Sjögren's syndrome, psoriasis, psoriatic arthritis, asthma, organ allograft rejection, graft vs. host disease (GVHD), lupus nephritis, IgA nephropathy, or scleroderma. 